Mutation Research 439 Ž1999. 277–285 Assessment of DNA damage in nurses handling antineoplastic drugs by the alkaline COMET assay ¨ ¨ Undeger ¨ ˘ a, Nurşen Başaran Ulku a a,) , Ayşe Kars b, Dicle Guç ¨ b Department of Pharmaceutical Toxicology, Faculty of Pharmacy, Hacettepe UniÕersity, Ankara 06100, Turkey b Institute of Oncology, Faculty of Medicine, Hacettepe UniÕersity, Ankara 06100, Turkey Received 18 August 1998; revised 27 October 1998; accepted 7 January 1999 Abstract The widespread use of chemotherapy in the treatment of cancer has led to anxiety about the possible hazards to staff involved in the preparation and administration of cytotoxic agents. Careless handling of antineoplastic drugs may lead to exposure in detectable amounts by means of chemical or biological methods in the body fluids or cell samples but the information about the mutagenic effects of these agents on nurses is limited and inconsistent. DNA damage in peripheral lymphocytes of 30 professional nurses employed in the oncology departments for at least 6 months were examined by the alkaline single cell gel electrophoresis, ‘COMET’ technique.The results were compared to that of 30 controls with comparable age, sex and smoking habits, not practising in the chemotherapy services. Work characteristics of the exposed nurses and the use of personal protective equipment were also investigated. The DNA damage observed in the lymphocytes of the nurses was significantly higher than the controls Ž p - 0.001.. The observed DNA damage was found to be significantly lower Ž p - 0.001. in nurses applying the necessary individual safety protections during their work. Cigarette smoking was not related to increases in DNA damage, also a significant association was not found between the duration of occupational exposure to antineoplastic drugs and the DNA damage. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Antineoplastic drug; DNA damage; Alkaline single cell gel electrophoresis; COMET 1. Introduction Antineoplastic drugs constitute a heterogenous group of chemicals that share an ability to inhibit tumour growth by disrupting cell division and killing actively growing cells. Many of the antineoplastic drugs in use are carcinogenic, mutagenic and teratogenic in laboratory animals. Moreover, during the ) Corresponding author. Tel.: q90-312-309-29-58; Fax: q90312-311-47-77 past decades, conclusive evidence has accumulated that treatment of malignant as well as non-malignant diseases with alkylating antineoplastic drugs carries a substantial risk of secondary malignancies, most notably acute non-lymphocytic leukemia w1x. Frequencies of sister chromatid exchanges ŽSCE. and chromosome aberrations ŽCA. were also significantly higher in patients undergoing chemotherapy compared to that in controls w2–4x. The widespread use of these mutagenic and carcinogenic drugs in the treatment of cancer has led to anxiety about possible hazards to staff who handle 1383-5718r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 3 - 5 7 1 8 Ž 9 9 . 0 0 0 0 2 - 9 278 ¨ Undeger ¨ ˘ et al.r Mutation Research 439 (1999) 277–285 U. them w5,6x. It is not known whether persons, especially nurses, from oncology departments involved in the preparation of these drugs are at risk although recent studies have suggested the possibility of an occupational health hazard among hospital personnel who handle cytotoxic drugs. Careless handling of such drugs may have been the cause of some subjective symptoms such as nausea, headache, dizziness, loss of hair among the personnel engaged in admixturing and administering antineoplastic drugs. Also, liver toxicity has been observed in nurses with long-term occupational contact with cytostatics w7,8x. Studies of occupational exposures have shown detectable levels of cytotoxic agents in the air of hospital units w9–11x. Several biomonitoring methods for the detection of occupational exposure to antineoplastic agents have been developed and validated. Methods like the determination of mutagenicity and thioethers in urine and cytogenetic methods such as the analysis of CAs, SCEs, and micronuclei in peripheral blood lymphocytes have been used frequently. Increased frequencies of SCEs and CAs w12–21x and higher levels of mutagens in urine concentrates w22–26x were found in hospital personnel handling cytotoxic drugs but negative findings have also been reported w27–32x. Subsequently, guidelines for health staff handling these drugs were issued by authorities in several countries, but great concern still exists about the possible adverse effects of occupational exposure. The alkaline single cell gel electrophoresis technique or COMET assay is a relatively new molecular assay which measures strand breaks incomplete excision repair sites, alkali labile sites and cross-linking, in individual cells. DNA damage is detected after electrophoresis of single cells embedded in agar where, under alkaline conditions the broken DNA strands move towards the anode forming a COMET w33x. The electric current pulls the charged DNA from the nucleus such that relaxed and broken DNA fragments migrate further. This assay has not yet been applied for the monitoring of genotoxic damage induced by antineoplastic drugs in oncology personnel. The objectives of this study were to apply the alkaline COMET assay to a group of nurses handling antineoplastic drugs and the unexposed controls and to investigate the potential hazards associated with the preparation and administration of these drugs, which are extensively used in cancer chemotherapy. 2. Materials and methods The chemicals used in these experiments were purchased from the following suppliers; normal melting agarose ŽNMA. and low melting agarose ŽLMA. from Boehringer Mannheim ŽGermany.; sodium chloride ŽNaCl. and sodium hydroxide ŽNaOH. from Merck Chemicals ŽDarmstadt, Germany.; dimethylsulfoxide ŽDMSO., ethidium bromide ŽEtBr., Triton X-100 and phosphate buffered saline ŽPBS. tablets from Sigma ŽSt. Louis, USA.; ethylenediamine tetraacetic acid disodium salt dihydrate ŽEDTA., Nlauroyl sarcosinate and Tris from ICN Biomedicals ŽAurora, OH, USA.. 2.1. Subjects The exposed group consisted of 30 professional nurses, currently employed in the oncology departments of different hospitals in the city of Ankara ŽTurkey., and who had been continuously involved in the preparation and administration of antineoplastic drugs for a period of 0.5–13 years. Thirty nurses, secretaries, and technicians of comparable age, sex, socio-economic life style and smoking habits, and with no history of antineoplastic drug exposure were used as the control group. The exposed group included 1 male and 29 female nurses ranging in age from 20 to 42 years Žmean 29 " 5 years., the control group consisted of 1 male and 29 female subjects ranging in age from 19–43 years Žmean 29 " 5 years.. A questionnaire designed to yield information on sex, age, occupational history, general health status, smoking and dietary habits, exposure to drugs and chemicals was used for each exposed and control subject. Work characteristics of the exposed nurses such as the use of protective equipment Žgloves, masks, gowns, eye glasses. and ventilation hoods and the existence of policies governing antineoplastic exposure were also investigated. 2.2. Blood sampling and lymphocyte preparations A 5 ml heparinized Ž50 unitsrmol sodium heparin. whole blood sample was taken by venepuncture ¨ Undeger ¨ ˘ et al.r Mutation Research 439 (1999) 277–285 U. from each individual at the end of the work week. Lymphocytes were isolated by Ficoll–Hypaque density gradient w34x and washed in PBS. Cell concentrations were adjusted to approximately 2 = 10 5rml in the buffer. 5–10 ml of the cells were suspended in 75 ml of 0.5% LMA for embedding on slides. Cells were checked for viability by trypan blue exclusion. 2.3. Slide preparation The basic alkaline technique of Singh et al. w35x, as further described by Anderson et al. w36x, was followed. Fully frosted microscopic slides ŽSurgipath, Winnipeg, Wanitoba. were each covered with 110 ml of 0.5% NMA at about 458C in Ca2q and Mg 2q free PBS. They were immediately covered with a large no. 1 coverslip and kept at room temperature for about 5 min to allow the agarose to solidify. This layer was used to promote the attachment of the second layer of 0.5% LMA. Approximately 10 000 cells were mixed with 75 ml of 0.5% LMA to from a cell suspension. After gently removing the coverslip, the cell suspension was rapidly pipetted onto the first agarose layer, spread using a coverslip, and maintained on an ice-cold flat tray for 5 min to solidify. After removal of the coverslip, the third layer of 0.5% LMA Ž75 ml. at 378C was added, spread using a coverslip, and again allowed to solidify on ice for 5 min. After removal of the coverslip the slides were immersed in cold lysing solution, Ž2.5 M NaCl, 100 mM Na 2 EDTA, 10 mM Tris, 1% sodium sarcosinate, pH 10., with 1% Triton-X 100 and 10% DMSO added just before use for a minimum of 1 h at 48C. 2.4. Electrophoresis The slides were removed from the lysing solution, drained and placed in a horizontal gel electrophoresis tank side by side, avoiding spaces and with the agarose ends facing each other, nearest the anode.The tank was filled with fresh electrophoresis solution Ž1 mM Na 2 EDTA and 300 mM NaOH, pH 13. to a level approximately 0.25 cm above the slides. Before electrophoresis, the slides were left in the solution for 20 min to allow the unwinding of the DNA and expression of alkali labile damage. Electrophoresis was conducted at a low temperature Ž48C. for 20 min using 24 V and adjusting the current to 300 mA by 279 raising or lowering the buffer level and using a compact power supply ŽPower Pack P 25 Biometra Analytic.. All of these steps were conducted under dimmed light Žtank was covered with a black cloth. to prevent the occurrence of additional DNA damage. After electrophoresis, the slides were taken out of the tank. Tris buffer Ž0.4 M Tris, pH 7.5. was added dropwise and gently to neutralize the excess alkali and the slides were allowed to sit for 5 min. The neutralizing procedure was repeated three times. 2.5. Staining To each slide, 65 ml EtBr Ž20 mgrml. was added. The slides were covered with a coverslip, placed in a humidified air-tight container to prevent drying of the gel, and analyzed within 3–4 h. 2.6. Slide scoring For visualization of DNA damage, slides were examined at a 1000 = magnification using a 100 = objective Žoil immersion. on a fluorescence microscope ŽZeiss, Germany.. Images of 200 randomly selected lymphocytes, i.e., 100 cells from each of two replicate slides, were analysed from each sample and the DNA damage was scored visually as described by Anderson et al. w36x. Comets are formed upon the principle of releasing damaged DNA from the core of the nucleus with electrophoresis, at low damage levels, stretching of attached strands of DNA is likely occur, and with increasing numbers of breaks, DNA pieces migrate freely into the tail of the comet. In the experiments, the cells were graded by eye into four categories on the basis of damage extent, i.e., undamaged Ž- 5%., slightly damaged Ž5–20%., damaged Ž20–40%. and highly damaged Ž) 40%.. Analysis was performed by one slide reader, thus minimising variability due to subjective scoring. 2.7. Statistical analysis Results are expressed as mean " SD, and the statistical comparison of the results from exposed and non exposed individuals, was performed using x 2 test. Student’s t-test was used to compare the grades of DNA damage in nurses and controls ac- ¨ Undeger ¨ ˘ et al.r Mutation Research 439 (1999) 277–285 U. 280 cording to smoking habits and also in nurses according to their individual protection. An analysis of linear regression was used to estimate the effects of the duration of occupational exposure of antineoplastic drugs on the DNA damage w37x. 3. Results According to the information from the nurses, the most frequently used cytostatic drugs were cyclophosphamide, methotrexate, 5-fluorouracil, adriamycin, bleomycin, cisplatin, vinblastine, vincristine, ifosfamide, and etoposide. Written guidelines or policies governing the handling of antineoplastic drugs were present in none of the hospitals. The work characteristics and the precautions taken during the exposure time and the number of cigarettes smoked of the oncology staff are shown in Table 1. There were 16 non-smokers and 14 smokers in the exposed and the control group. The average cigarette consumption of smokers in both groups was nearly 15 cigarettes per day. Nearly half of the nurses Ž n: 13. handling antineoplastic drugs wore gloves, masks and protective gowns with a closed front and long cuffed sleeves. The appropriate ventilatory devices Table 1 The work characteristics and the cigarette consumption of the oncology nurses Subject no. Age Number of cigarettes smokedrday BI˙ PT 35 31 4.0 0.5 Ha N ¨ HO KA 34 35 q Ž10rday. – q Ž10rday. q Ž10rday. 2.0 1.0 N N ¨ HO LP NM ŞA 30 27 28 30 – – – q Ž5rday. 12.0 1.0 3.0 2.0 N N N N ¨ AO DG AE SK 28 25 30 25 – q Ž10rday. – q Ž8rday. 3.0 5.5 13.0 4.0 N N Ha G, SM, H, PG ¨ ZU LD CK KS EG 24 33 25 35 25 – q Ž5rday. – – – 6.0 2.0 4.0 3.5 4.0 N N N Ha G, SM, H, PG ¨ ZO NÇ FT SB MA FG 25 25 40 35 26 30 4.0 6.5 1.5 6.0 3.5 6.0 G, SM, H, PG Ha G, SM, H, PG G, SM, H, PG G, SM, H, PG G, SM, H, PG ¨ MO EY AA 42 23 23 – – q Ž4rday. q Ž15rday. q Ž15rday. – q Ž30rday. q Ž20rday. q Ž20rday. 7.0 0.7 0.4 G, SM, H, PG G, SM, H, PG N İB DS MK SE 23 22 28 20 – q Ž20rday. – – 4.8 1.0 2.0 0.5 G, SM, H, PG G, SM, H, PG G, SM, H, PG G, SM, H, PG G: gloves; SM: surgical mask; PG: protective gowns; H: hood; N: none. a Occasionally use. Handling time of antineoplastics Žyears. Type of protection ¨ Undeger ¨ ˘ et al.r Mutation Research 439 (1999) 277–285 U. 281 Table 2 Grades of DNA damage in peripheral lymphocytes of the nurses and the controls Groups Nurses Grades of DNA damage Groups Controls Grades of DNA damage Undamaged Slightly damaged Damaged Highly damaged Undamaged Slightly damaged Damaged Highly damaged BI˙ PT 118 162 48 19 17 15 17 4 200 200 MB MB 119 197 46 2 20 0 15 1 200 200 ¨ HO KA 28 91 76 57 78 40 18 12 200 200 TK GÇ 153 174 31 6 6 12 10 8 200 200 ¨ HO LP NM ŞA 62 52 94 156 47 56 79 34 65 58 26 6 26 34 1 4 200 200 200 200 HS SB BG TB 170 197 187 174 18 0 10 20 6 0 1 0 6 3 2 6 200 200 200 200 ¨ AO DG AE SK 96 111 159 142 76 60 28 30 14 14 11 15 14 15 1 13 200 200 200 200 NK HS BA HY 172 198 174 157 20 2 15 34 3 0 7 1 5 0 4 8 200 200 200 200 ¨ ZU 107 72 15 6 200 ¨¨ UU 181 12 0 7 200 LD CK 108 126 66 28 12 22 14 24 200 200 ¨ BO YA 170 193 18 4 4 2 8 1 200 200 KS EG 170 120 14 46 8 8 8 26 200 200 ¨ AO PK 185 195 14 2 1 2 0 1 200 200 ¨ ZO NÇ FT SB MA FG 173 142 185 177 154 173 22 20 8 13 36 26 1 22 2 6 6 0 4 16 5 4 4 1 200 200 200 200 200 200 MY SA BU HD SK OO 168 176 160 190 192 124 14 10 38 10 6 46 8 4 2 0 0 24 8 10 0 0 2 6 198 200 200 200 200 200 ¨ MO EY AA 163 168 167 28 26 30 7 4 2 2 2 1 200 200 200 ¨ KO GŞ AK 164 179 188 23 21 9 4 0 0 9 0 3 200 200 200 İB DS MK 188 183 177 11 8 18 1 5 4 0 4 1 200 200 200 PA GS NG 183 168 196 4 17 0 10 5 1 3 10 3 200 200 200 ¨ EU Mean " SD 197 176.0 " 19.6 3 15.2 " 13.0 0 4.1 " 5.9 0 5.7 " 6.1 SE 177 Mean " 137.6 " SD 42.9 20 36.7 " 22.0 2 16.2 " 19.5 1 9.4 " 9.2 Total 200 6000 Table 3 Grades of DNA damage in peripheral lymphocytes of the nurses and the controls Groups Grades of DNA damage Undamaged No. Nurses 4129 Controls 5281 x 2 s 674.8, p s 0.0000 Slightly damaged Damaged % No. % No. 68.8 88.0 1102 455 18.4 7.6 486 123 Highly damaged Total % No. % No. 8.1 2.1 282 139 4.7 2.3 6000 5998 Total 200 5998 ¨ Undeger ¨ ˘ et al.r Mutation Research 439 (1999) 277–285 U. 282 Table 4 Grades of DNA damage in nurses and controls according to smoking habits Groups n Grades of damage Undamaged Slightly damaged Damaged Highly damaged 8.2 " 6.2 1 10.4 " 11.3 3 9.4 " 9.2 6 Nurses Smokers Non-smokers Total 14 16 30 139.4 " 43.9 1 136.1 " 43.4 5 137.6 " 42.9 7 37.1 " 21.4 2 36.4 " 23.15 36.7 " 22.0 7 15.3 " 20.51 17.0 " 19.2 4 16.2 " 19.5 6 Controls Smokers Non-smokers Total 14 16 30 170.4 " 20.2 180.9 " 18.4 176.0 " 19.7 20.1 " 13.2 10.9 " 11.4 15.2 " 13.0 3.9 " 5.8 4.3 " 6.2 4.1 " 5.9 5.6 " 4.8 3.8 " 3.0 4.6 " 4.0 1 p - 0.05 DNA damage in smoking nurses compared with smoking controls. 2 p - 0.01 DNA damage in smoking nurses compared with smoking controls. 3 p - 0.05 DNA damage in non-smoking nurses compared with non-smoking controls. 4 p - 0.01 DNA damage in non-smoking nurses compared with non-smoking controls. 5 p - 0.001 DNA damage in non-smoking nurses compared with non-smoking controls. 6 p - 0.01 DNA damage in nurses compared with controls. 7p - 0.001 DNA damage in nurses compared with controls. for preparing the drugs were in place for these nurses, but the other 17 nurses had not taken the safety individual precautions and did not even wear gloves. Although 4 of the 17 nurses had safety cabinet facilities for preparing the antineoplastic drugs, they did not use them frequently. None of the nurses wore safety glasses. The duration of antineoplastic drug handling varied from 6 months to 13 years but the exact number of hours that the drugs were handled per day could not be assessed accurately. In all the alkaline comet assays viability generally exceeded 90%, and the results of the grades of DNA damage in peripheral lymphocytes of the nurses and the controls are summarized in Tables 2 and 3. A significant increase Ž x 2 s 674.8, p s 0.0000. in the DNA damage Ž- 5%. was observed in the oncology staff involved in the preparation and administration of cytotoxic drugs. The number of lymphocytes without DNA damage was significantly higher in the unexposed controls compared to that the nurses Ž p - 0.001.. The correlations between the duration of exposure and the grades of DNA damage; i.e., undamaged Ž- %5., slightly damaged Ž5–20%., damaged Ž20–40%. and highly damaged Ž) 40%. were r s y0.08 p ) 0.05, y0.16 p ) 0.05, 0.12 p ) 0.05 and 0.11 p ) 0.05, respectively. A significant association could not be found between the DNA damage and the length of exposure. Cigarette smoking was also not related to DNA damage observed in the lymphocytes of the nurses since no additional DNA damage was observed when the smoking and the non-smoking nurses were compared ŽTable 4.. As shown in Table 5, 13 nurses who had taken the necessary individual safety precautions have less DNA damage compared to 17 who had not. The increases in the number of slightly damaged, damaged and highly damaged cells were significant in nurses without protection Ž0.002 p - 0.01, 0.003 Table 5 DNA damage in nurses according to their individual protection Nurses Without safety protection With safety protection 1 p - 0.05. 2 p - 0.01. n 17 13 Grade of damage Undamaged Slightly damaged Damaged Highly damaged 114.6 " 42.0 167.7 " 19.2 1 47.6 " 22.1 22.5 " 11.2 2 25.0 " 22.1 4.7 " 4.0 2 12.6 " 9.5 5.2 " 7.11 ¨ Undeger ¨ ˘ et al.r Mutation Research 439 (1999) 277–285 U. p - 0.01, 0.014 p - 0.05, respectively. whereas the number of undamaged cells were increased in nurses with protection Ž0.019 p - 0.05.. 4. Discussion The main routes of occupational exposure for the personnel handling antineoplastic drugs appear to be inhalation and percutaneous absorption since contamination may occur during the reconstitution of parenteral antineoplastics, during the normal process of excess-drug disposal or as a result of vial leakage, or accidental spill w37,38x. Polyethylene glove material was found to be permeable and contamination of and permeation through latex gloves were found for cyclophosphamide, 5-fluorouracil, and methotrexate w39,40x. Evidence that measurable concentrations of cytotoxic agents can be detected in the vicinity of the handling sites, if unprotected is present. Detectable concentrations of cyclophosphamide were measured primarily on horizontal surfaces of the uncontrolled hospital environment where a biological safety cabinet was not in use w6,9–11x. Positive wipe samples were found at the nurses station and in the short-term treatment area w10x. Therefore, effective safety precautions are necessary to minimize all potential exposure possibilities and since possible hazards of exposure to health personnel in several studies are reported, extensive improvements in the handling practices of antineoplastic drugs have been performed in the hospitals of many developed countries. In 1986, the US Occupational Safety and Health Administration ŽOSHA. published guidelines for handling antineoplastics that recommended protective clothing and equipment for mixing and administering the drugs, cleaning up spills, and handling excreta of cancer patients who have received chemotherapy during the previous 48 h w41x. The examined nurses in this study had not been aware of the adequate recommendations for the management of antineoplastic drugs, and they had handled them for a long period of time without protection. There were no written guidelines or policies governing the handling of these drugs in the oncology departments of the hospitals. Use of safety precautions by nearly half of the nurses appeared to have improved over the last years. Although the DNA damage observed 283 in the oncology nurses was significantly higher Ž p 0.001. than the controls, the damage in 13 nurses who used gloves, gowns and safety cabinets for drug preparation was less when compared to that in 17 nurses who did not protect themselves. The high DNA damage observed in the 13 nurses may be due to early exposures when no special attention was paid to careful handling of the drugs or use of protection might have been neglected from time to time. From the workers protection point of view, it is important to monitor the possible risk. Results with biological monitoring or cytogenetic surveillance methods have given both positive and negative results of mutagenicity among nurses handling antineoplastic drugs w12–21x w22–32x w42x. The present study is the first study by the alkaline COMET assay showing that nurses having an increase in DNA damage related to their work are exposed to at least one or two antineoplastic drug. The results were compared to those from a group of nurses, secretaries, and technicians working under similar conditions as the first group but not handling cytostatic drugs. Matched control groups were used because of possible confounders like age, sex, smoking and exposure to X-rays and chemicals. The results are consistent with the results of Fuchs et al. w43x, who measured the amount of DNA single strand breaks and alkali labile sites in the peripheral mononuclear blood cells of 91 nurses handling antineoplastic drugs in four German hospitals and compared them with 54 controls using the alkaline elution method. A 50% higher level of DNA strand breaks was detected in nurses not using recommended safety precautions but in nurses, using adequate safety equipment, no significant differences were found. Our results are also consistent with the studies of Bayhan et al. w44x, and Şardaş et al. w14x, who reported on the possible risk for the oncology nurses not having adequate safety requirements in this country. Şardaş et al. w14x examined the SCE frequency of 23 nurses working in the haematology and oncology departments and a significant increase in SCE frequency was observed in nurses as compared to a group of 50 unexposed controls. In the other study, a significant increase in the urinary thioether excretion was found in the oncology nurses w44x. In both studies w14,44x, quite differently from ours, smoking nurses handling antineoplastic drugs 284 ¨ Undeger ¨ ˘ et al.r Mutation Research 439 (1999) 277–285 U. were found to have a greater risk compared their non-smoking colleagues. We did not observe additional DNA damage in smoking nurses. 5. Conclusion In conclusion, the results of this study demonstrate the possibility of genetic risk to individuals that handle antineoplastic drugs if safety requirements are not fulfilled and emphasize the necessity and the importance of protective measures and safety rules. Use of adequate hoods, gloves and garment would provide appropriate working conditions. Acknowledgements This study could not have been carried out without the collaboration with the occupational personnel of the oncology units of the hospitals of Ankara, and it was supported by Grant SBAG-1685 from the Scientific and Technical Research Council of Turkey ¨ ˙ ŽTUBITAK .. References w1x IARC, Monograps on the evaluation of carcinogenic risk of chemicals to humans, in: Some Antineoplastic and Immunosuppressive Agents, Vol. 26, International Agency for Research on Cancer, Lyon, France, 1981, 441 pp. w2x J. Musilova, K. Michalova, J. Urban, Sister-chromatid exchanges and chromosomal breakage in patients treated with cytostatics, Mutat. Res. 67 Ž1979. 289–294. w3x E. Gebhart, J. Losing, F. Wopfner, Chromosome studies on ¨ lymphocytes of patients under cytostatic therapy: I. Conventional chromosome studies in cytostatic interval therapy, Hum. Genet. 55 Ž1980. 53–63. w4x M.M. Aronson, R.C. Miller, R.B. Hill, W.W. Nichols, A.T. Meadows, Acute and long term cytogenetic effects of treatment of childhood cancer: sister-chromatid exchanges and chromosome aberrations, Mutat. Res. 92 Ž1982. 291–307. w5x M. Sorsa, K. Hemminki, H. Vainio, Occupational exposure to anticancer drugs—potential and real hazards, Mutat. Res. 154 Ž1985. 135–149. w6x M. Sorsa, D. Anderson, Monitoring of occupational exposure to cytostatic anticancer agents, Mutat. Res. 355 Ž1996. 253– 261. w7x M. Tortorici, Precautions followed by personnel involved with the preparation of parenteral antineoplastic medications, Hosp. Pharm. 15 Ž1980. 293–301. w8x C.F. Ladik, G.P. Stoehr, M.A. Maur, Precautionary measures in the preparation of antineoplastics, Am. J. Hosp. Med. 37 Ž1980. 1184–1186. w9x L. Pyy, M. Sorsa, E. Hakala, Ambient monitoring of cyclophosphamide in manufacture and hospitals, Am. Ind. Hyg. Assoc. J. 49 Ž1988. 314–317. w10x J.J. McDevitt, P.S.J. Lees, M.A. McDiarmid, Exposure of hospital pharmacists and nurses to antineoplastic agents, J. Occup. Med. 35 Ž1993. 57–60. w11x P.J.M. Sessink, J.L. Timmersmans, R.B.M. Anzion, R.P. Bos, Assessment of occupational exposure of pharmaceutical plant workers to 5-fluorouracil, J. Occup. Med. 36 Ž1994. 79–83. w12x H. Norppa, M. Sorsa, H. Vainio, P. Grohn, ¨ E. Hoinonen, L. Holsti, E. Nordman, Increased sister chromatid exchange frequencies in lymphocytes of nurses handling cytostatic drugs, Scand. J. Work Environ. Health 67 Ž1980. 229–301. w13x H. Wasvik, O. Klepp, A. Brøgger, Chromosome analyses of nurses handling cytostatic agents, Cancer Treat. Rep. 65 Ž1981. 607–610. w14x S. Şardaş, S. Gok, ¨ A.E. Karakaya, Sister chromatid exchanges in lymphocytes of nurses handling antineoplastic drugs, Toxicol. Lett. 55 Ž1991. 311–315. w15x E.M. Goloni-Bertollo, E.H. Tajara, A.J. Manzato, M. Varella-Garcia, Sister chromatid exchanges and chromosome aberrations in lymphocytes of nurses handling antineoplastic drugs, Int. J. Cancer 50 Ž1992. 341–344. w16x E. Nikula, K. Kiviniitty, J. Leisti, P.J. Taskinen, Chromosome aberrations in lymphocytes of nurses handling cytostatic agents, Scand. J. Work Environ. Health 10 Ž1984. 71–74. w17x J. Pohlova, M. Cerna, P. Rossner, Chromosomal aberrations, ¨ SCE and urine mutagenicity in workers occupationally exposed to cytostatic drugs, Mutat. Res. 174 Ž1986. 213–217. w18x U. Oestreicher, G. Stephan, M. Glatzel, Chromosome and SCE analysis in peripheral lymphocytes of persons occupationally exposed to cytostatic drugs handled with and without use of safety covers, Mutat. Res. 241 Ž1990. 177–271. w19x A. Krepinsky, D.W. Bryant, L. Davison, B. Young, J. Heddle, D.R. McCalla, G. Douglas, K. Michalko, Comparison of three assays for genetic effects of antineoplastic drugs on cancer patients and their nurses, Environ. Mol. Mutagen. 15 Ž1990. 83–92. w20x P.J.M. Sessink, M. Cerna, P. Rossner, A. Pastorcova, H. ¨ Bavarova, K. Frankova, R.B.M. Anzion, R.P. Bos, Urinary cyclophosphamide excretion and chromosomal aberrations in peripheral blood lymphocytes after occupational exposure to antineoplastic agents, Mutat. Res. 309 Ž1994. 193–199. w21x W.A. Anwar, Assessment of cytogenetic changes in human populations at risk in Egypt, Mutat. Res. 313 Ž1994. 183–191. w22x K. Falck, P. Grohn, ¨ M. Sorsa, H. Vainio, E. Heinonen, L.R. Holsti, Mutagenicity in urine of nurses handling cytostatic drugs, Lancet i Ž1979. 1250–1251. w23x R.W. Anderson, W.H. Puckett, W.J. Dana, T.V. Nyugen, J.C. Theiss, T.S. Matney, Risk of handling injectable antineoplastic agents, Am. J. Hosp. Pharm. 39 Ž1982. 1881–1887. w24x R.P. Bos, A.O. Leenaars, J.L.G. Theuws, P.T. Henderson, ¨ Undeger ¨ ˘ et al.r Mutation Research 439 (1999) 277–285 U. w25x w26x w27x w28x w29x w30x w31x w32x w33x w34x Mutagenicity of urine from nurses handling cytostatic drugs, influence of smoking, Int. Arch. Occup. Environ. Health 50 Ž1982. 359–369. T.V. Nyugen, J.C. Theiss, T.S. Matney, Exposure of pharmacy personnel to mutagenic antineoplastic drugs, Cancer Res. 42 Ž1982. 4792–4796. S. Venitt, C. Crofton-Sleigh, J. Hunt, V. Speechley, K. Briggs, Monitoring exposure of nursing and pharmacy personnel to cytotoxic drugs: urinary mutation assays and urinary platinum as markers of absorbtion, Lancet i Ž1984. 74–77. B. Kolmodin-Hedman, P. Hartvig, M. Sorsa, K. Falck, Occupational handling of cytostatic drugs, Arch. Toxicol. 54 Ž1983. 25–33. R. Barale, G. Sozzi, P. Toniolo, O. Borghi, D. Reali, N. Loprieno, G. Della Porta, Sister-chromatid exchanges in lymphocytes and mutagenicity in urine of nurses handling cytostatic drugs, Mutat. Res. 157 Ž1985. 235–240. M. Sorsa, L. Pyy, S. Salomaa, L. Nylund, J.W. Yager, Biological and environmental monitoring of occupational exposure to cyclophosphamide in industry and hospitals, Mutat. Res. 204 Ž1988. 465–479. I. Strucker, A. Hirsch, T. Doloy, I. Bastie-Sigeac, D. Hemon, Urine mutagenicity, chromosomal abnormalities and sister chromatid exchanges in lymphocytes of nurses handling cytostatic drugs, Int. Arch Occup. Environ. Health 57 Ž1986. 195–205. D. Poyen, M.P. DeMeo, ´ A. Botta, J. Gouvernet, G. Dumenil, ´ Handling of cytostatic drugs and urine mutagenesis, Int. Arch. Occup. Environ. Health 61 Ž1988. 183–188. J. Cooke, J. williams, R.J. Morgan, P. Cooke, R.T. Calvert, Use of cytogenetic methods to determine mutagenic changes in the blood of pharmacy personnel and nurses who handle cytotoxic agents, Am. J. Hosp. Pharm. 48 Ž1991. 1199–1205. D.W. Fairbairn, P.L. Olive, K.L. O’Neill, The COMET assay: a comprehensive review, Mutat. Res. 339 Ž1995. 37–59. A. Boyum, Isolation of lymphocytes, granulocytes, and macrophages, Scand. J. Immunol. 5 Ž1976. 9–15. 285 w35x N.P. Singh, M.T. McCoy, R.R. Tice, E.L. Schneider, A simple technique for quantitation of low levels of DNA damage in individual cells, Exp. Cell. Res. 175, 184–191. w36x D. Anderson, T.-W. Yu, B.J. Philips, P. Schmezer, The effect of various antioxidants and other modifying agents on oxygen radical generated DNA damage in human lymphocytes in the COMET assay, Mutat. Res. 307 Ž1994. 261–271. w37x M. Hirst, D.G. Mills, S. Tse, L. Levin, D.F. White, Occupational exposure to cyclophosphamide, Lancet i Ž1984. 186– 188. w38x J.M. Stellman, Assessment of potential exposure to antineoplastic agents in the health care setting, Prev. Med. 13 Ž1984. 245–255. w39x J.L. Laidlaw, T.H. Connor, J.C. Theiss, R.W. Anderson, T.S. Matney, Permeability of latex and polyvinyl chloride gloves to 20 antineoplastic drugs, Am. J. Hosp. Pharm. 41 Ž1984. 2618–2623. w40x P.J.M. Sessink, M.C.A. Van de Kerkhof, R.B.M. Anzion, J. Noordhoek, R.P. Bos, Environmental contamination and assessment of exposure to antineoplastic agents by determination of cyclophosphamide in urine of exposed pharmacy technicians: is skin absorption an important exposure route?, Arch. Environ. Health 49 Ž1994. 165–169. w41x OSHA work-practice guidelines for personnel dealing with cytotoxic Žantineoplastic. drugs, Am. J. Hosp. Pharm. 43 Ž1986. 1193–1204. w42x S. Roth, H. Norppa, H. Jarventaus, P. Kyyronen, M. Ahonen, ¨ ¨ ¨ J. Lehtomaki, H. Sainio, M. Sorsa, Analysis of chromosomal aberrations, sister chromatid exchanges and micronuclei in peripheral lymphocytes of pharmacists before and after working with cytostatic drugs, Mutat. Res. 325 Ž1994. 157–162. w43x J. Fuchs, J.G. Hengstler, D. Jung, J. Hiltle Konietzko, F. Oesch, DNA damage in nurses handling anti-neoplastic agents, Mutat. Res. 342 Ž1995. 17–23. w44x A. Bayhan, S. Burgaz, A.E. Karakaya, Urinary thioether excretion in nurses at an oncologic department, J. Clin. Pharm. Ther. 12 Ž1987. 303–306.