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Mutagenesis Advance Access originally published online on July 21, 2005
Mutagenesis 2005 20(5):351-357; doi:10.1093/mutage/gei048
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© The Author 2005. Published by Oxford University Press on behalf of the UK Environmental Mutagen Society. All rights reserved. For permissions, please email: journals.permissions@oupjournals.org

Investigating genetic damage in workers occupationally exposed to methotrexate using three genetic end-points

Hongping Deng1, Meibian Zhang1, Jiliang He1,2,*, Wei Wu1, Lifen Jin1, Wei Zheng1, Jianlin Lou1 and Baohong Wang1

1Zhejiang University, Medical College, Institute of Occupational and Environmental Health, Hangzhou 310006, Zhejiang, People's Republic of China and 2Medical College of Jiaxing University, Jiaxing 314001, Zhejiang, People's Republic of China


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 
Genetic damage in workers occupationally exposed to an antineoplastic drug was studied using the micronucleus (MN) test, the comet assay, the hprt gene mutation assay and the TCR gene mutation assay. The subjects were divided into two groups: (i) 21 workers from a plant producing methotrexate (MTX); (ii) 21 controls were matched according to age, gender and smoking. Fresh blood samples were collected from the workers and controls. The results of the MN test showed that the mean micronuclei rate (MNR) and mean micronucleated cell rate (MCR) in workers were 10.10 ± 0.95{per thousand} and 8.05 ± 0.75{per thousand}, respectively, which were significantly higher than those (5.48 ± 0.82{per thousand} and 4.38 ± 0.58{per thousand}) in controls (P < 0.01). It was found in the comet assay that the mean tail length (MTL) of workers and controls were 1.30 ± 0.06 µm and 0.07 ± 0.01 µm, respectively. There was a significant difference between workers and controls for MTL (P < 0.01), but the difference between the mean tail moment (MTM, 0.23 ± 0.03) of workers and MTM (0.17 ± 0.04) of controls was not significant (P > 0.05). The results of hprt gene mutation assay showed that the average mutation frequency (Mf-hprt) of hprt in workers was 1.00 ± 0.02{per thousand}, which was significantly higher than that (0.86 ± 0.01{per thousand}) in controls (P < 0.01). Meanwhile, the results of TCR gene mutation assay indicated that Mfs-TCR gene mutation frequencies of workers and controls were 6.87 ± 0.52 x 10–4 and 1.67 ± 0.14 x 10–4, respectively, which were significantly different (P < 0.01). The results of our experiment suggest that genetic damage is detectable in the 21 workers occupationally exposed to methotrexate.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Conclusion
 References
 
The widespread use of chemotherapy to treat cancer has led to concerns about the possible hazards to staff and workers involved in the preparation, administration and production of cytotoxic agents. Antineoplastic drugs constitute a heterogenous group of chemicals that share an ability to inhibit cancer growth by killing actively growing cells and disrupting cell division. However, many antineoplastic drugs have been shown to be carcinogenic, mutagenic and teratogenic in experimental animals and in vitro test systems. Antineoplastic agents include cytostatic drugs, hormones, antibiotics and other supportive therapy. Cytostatic drugs can further be divided into alkylating agents, antimetabolites and mitotic inhibitors, free radical generators and topoisomerase II inhibitors. Since the drugs have different mechanisms of action to destroy the malignant growth of cells, combinative chemotherapy is most frequently used. Most epidemiological studies of occupational exposure to antineoplastic agents were carried out in people employed in the preparation and administration of the drugs to patients and in nursing patients so the persons commonly were occupationally exposed to the mixed antineoplastic drugs (1GoGoGoGoGoGoGoGoGoGoGoGoGoGoGo–16Go). In these studies, the methods used to detect cytogenetic damage are sister chromatid exchange (SCE) test, chromosomal aberration (CA) test, micronucleus (MN) test, comet assay and gene mutation test.

Methotrexate (MTX) is an antineoplastic drug with the action mode of antimetabolites. Although the evidence for human carcinogenicity of MTX is considered inadequate by IARC, MTX can induce SCEs, CAs and MN, the chemical is clearly active at the chromosomal level in rodent and in human cells in vitro (2Go,17Go). Recently, some investigations showed that MTX could induce increased frequencies of micronulei and CAs in both rats and humans (18GoGo–20Go). Keshava et al. (21Go,22Go) reported that MTX could induce a significant increase in percent micronucleated binucleated cells (MNBNs) and percent aberrant cells (Abs) in V79 cells. In order to determine whether the genetic damage appears in the workers only occupationally exposed to MTX, in the present investigation three genetic endpoints, i.e. chromosomal damage (MN test), gene mutation (hprt gene mutation assay and TCR gene mutation assay) and DNA damage (comet assay) were investigated.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Conclusion
 References
 
Subjects and blood sample collection
The peripheral blood was from 21 controls and 21 workers only occupationally exposed to MTX. The workers group consisted of 11 males and 10 females from 19 to 50 years old (mean age, 35 years old), who were from a workshop producing MTX. The average ages of male workers and female workers are 32 and 38 years old, respectively. The difference between male workers' average age and female workers' average age is not significant (P = 0.165). The controls were matched with workers on the basis of age (from 21 to 55 years old, mean age, 37 years old), gender and smoking. The general situation of workers and controls is listed in Table I, and shows no significant difference between workers and controls for gender, age and smoking habits.


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  		Table I.. General description of 21 workers and 21 controls

 
The area of the workplace is only about 200 m2 without good ventilating equipment, and 20 workers used protective measures (gloves and masks). The individual procedures include: cleaning, producing, packing, testing and compounding. The exposure situation for workers is similar.

Cytokinesis-block micronucleus (CBMN) assay
The MN test was conducted according to the method of Fenech and Morley (23Go) and Lou et al. (24Go). Briefly, 0.5 ml whole blood was added to 4.5 ml RPMI 1640 (GIBCO) containing 20% fetal calf serum, 0.2 mg/ml phytohaemagglutinin-M (PHA-M, GIBCO). Two independent cultures were incubated at 37°C for 72 h. Cytochalasin B (Sigma) 4.5 µg/ml was added to each culture at 28 h before harvesting cells. The cultures were harvested by centrifugation after 72 h. Cells were then processed on the basis of the method described by Fenech (23Go) and modified to enable the use of whole blood culture. The lymphocytes were subjected to mild hypotonic treatment (0.075 M KCl) for 5 min, then fixed in fresh fixative solution (methanol:acetic acid, 3:1) for 20 min, this fixation step was repeated twice after a 20 min storage at 4°C. The cells were smeared on microscope slides, air-dried, and stained for 10 min with 10% pH 6.8 Giemsa solution. One thousand binucleated lymphocytes (500 cells per culture) were scored under light microscopy (magnification x400). The micronuclei and micronucleated cells were detected according to the criteria described by Fenech (25Go). All the slides were examined by the same person. Number of micronucleated cells (i.e. micronucleated cell rate, MCR) and number of micronuclei (i.e. micronucleus rate, MNR) per 1000 binucleated lymphocytes served as the indicators. Meanwhile, nuclear division index (NDI) was calculated on the basis of the following formula (24Go,26Go):

Comet assay
Human lymphocytes were isolated with the procedure described by Lou et al. (24Go) and Zhang et al. (26Go), and were resuspended in phosphate-buffered saline (PBS). Following isolation, the cells were mixed with 0.4% Trypan blue solution. After 15 min, cells were counted and checked for viability. The remaining cells were immediately used for single cell gel electrophoresis. The assay was performed basically according to Singh et al. (27Go). Roughened slides were cleaned with 100% ethanol and air-dried. Two solutions, 0.5% normal melting agarose and 0.5% low melting agarose, were prepared in Ca2+, Mg2+-free PBS. 100 µl of normal melting agarose was used for the first layer, while 75 µl low melting agarose +10 µl PBS cell suspension (10000 cells) was used for the second layer. Finally, the third layer of 75 µl low melting agarose was added. Slides were immersed in freshly prepared lysis solution (1% sodium sarcosinate, 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris–HCl, pH 10, 1% Triton X-100 and 10% dimethyl sulfoxide) at 4°C for 1 h. Then slides were placed in a horizontal electrophoresis unit covered with fresh buffer (1 mM Na2EDTA, 300 mM NaOH, pH 13) for 20 min to allow DNA unwinding and expression of alkali-labile sites. Electrophoresis was conducted for 20 min at 20 V and 300 mA. The above treatments were performed in an ice bath. Subsequently, slides were washed gently three times to remove alkali and detergent in a neutralization buffer (0.4 M Tris–HCl, pH 7.5). Each slide was stained with 50 µl ethidium bromide (20 µg/ml). All the above steps were conducted under yellow light to prevent additional DNA damage.

Observations were made at x400 magnification using a fluorescence microscope (Olympus, BX51) equipped with a 530 nm excitation filter, a 590 nm emission filter, a camera (Olympus, DP50) and a computer-based image analysis system (Media Cybernetics, Inc., USA). The data were based on 50 randomly selected cells per sample, i.e. 25 cells were from each of the two replicate slides. The mean tail length (MTL) and mean tail moment (MTM) served as the indicators.

The hprt gene mutation assay
The hprt gene mutation assay was performed according to the description by Cao et al. (28Go). Two sets of cultures were prepared, each set of culture contained 0.5 ml heparinized blood and 4.5 ml RPMI 1640 with 20% fetal calf serum and 0.2 mg/ml PHA-M. One set of culture was added with 0.2 mM 6-thioguanine (Sigma). At 33 h of incubation, Cytochalasin B (the final concentration, 4.5 µg/ml) was put into two sets of cultures. At 72 h of incubation, the lymphocytes were harvested by centrifugation and fixed with methanol: acetic acid (3:1). The slides were prepared and stained with 10% pH 6.8 Giemsa solution. The binucleated and multinucleated cells per 1000 lymphocytes in two sets of cultures were scored under light microscopy (magnification x400). Mutant frequency of hprt gene (Mf-hprt) was calculated with the following formula:


{gei048in1}

The TCR gene mutation assay
The TCR gene mutation was detected using flow cytometric assay. The assay was conducted basically according to Kyoizumi et al. (29Go). The frequency of TCR/CD3 mutant cells (TCR Mf) in CD4+ cells was measured by two-color flow cytometry. Briefly, the peripheral blood mononuclear cells (>2 x 105) were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD4 MoAb and phycoerythrin (PE)-labeled anti-CD3 MoAb obtained from Becton Dickinson Immunocytometry System. After being washed, cell suspensions were analyzed by Coulter EPICS XL (Coulter Co., Hialeah, FL, USA) with System software. The lymphocyte fraction was gated by forward and right-angle light scatter, and >2 x 105 events were acquired in dual parameter (FITC and PE) correlated mode. The mutant window was set to the region where the surface CD3 level was <0.04 of that for normal CD4+ cells. The left and right limits of CD4 FITC fluorescence for the mutant window were set, respectively, at 0.5 and 2 times the mode of FITC fluorescence intensity for normal CD3+4+ T cells. The Mf was calculated as the number of events in the mutant cell window divided by the total number of CD4+ T cells in the flow cytogram.

Statistical analysis
It was found that the data of MCR, MNR, MTM and Mf-hprt gene were homoscedastic, so the statistical analysis was carried out using the t-test. Meanwhile, the data of MTL, NDI and Mf-TCR gene were heteroscedastic so that the statistical analysis was carried out using the Wilcoxon's rank-sum test. Kendall's test was used to analyze the correlation between exposure years and six parameters. Statistical analyses were performed with SPSS 11.0 program for windows.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Conclusion
 References
 
The results of MN test in 21 workers and 21 controls
The results of CBMN test in 21 workers and 21 controls are presented in Table II. The ranges of MCRs in workers and controls were 4–17{per thousand} and 1–10{per thousand}, respectively, while the mean MCRs of workers and controls were 8.05 ± 0.75{per thousand} and 4.38 ± 0.58{per thousand}, respectively. The mean MCR of workers was significantly higher than that of controls (P < 0.01). The ranges of MNRs of workers and controls were 4–20{per thousand} and 1–15{per thousand}, respectively. The average MNR of workers was 10.10 ± 0.95{per thousand}, which was significantly higher than that (5.48 ± 0.82{per thousand}) of controls (P < 0.01). The distribution of micronuclei per cell and the NDIs of workers and controls were listed in Table II. The ranges of NDIs of workers and controls were 1.74–2.04 and 1.61–2.37, respectively. The average NDI of workers was 1.87 ± 0.02, which was less than that (2.00 ± 0.05) of controls (P < 0.05).


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  		Table II.. The results of MN test in 21 workers and 21 controls

 
The results of comet assay in 21 workers and 21 controls
Table III displays the results of comet assay with MTL and MTM in workers and controls. In the comet assay, DNA damage of lymphocytes was shown by tail length and tail moment. The ranges of the MTL and the MTM were 0.75–2.01 µm and 0.07–0.58 in workers, respectively, while the MTL and the MTM were 0.67–0.82 µm and 0.08–0.90 in controls, respectively. The average MTL and MTM of workers were 1.30 ± 0.06 µm and 0.23 ± 0.03, respectively. The average MTL and MTM of controls were 0.07 ± 0.01 µm and 0.17 ± 0.04, respectively. There was a significant difference between workers and controls for MTL (P < 0.01), but the difference between workers and controls for MTM was not significant (P > 0.05).


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  		Table III.. The results of TCR gene mutation test, hprt gene mutation test, comet assay in 21 workers and 21 controls

 
The results of hprt gene mutation assay in 21 workers and 21 controls
Mfs-hprt for workers and controls are shown in Table III. The ranges of Mf-hprt for workers and controls were 0.89–1.20 and 0.76–0.93{per thousand}, respectively. The average Mfs-hprt for workers and controls were 1.00 ± 0.02{per thousand} and 0.86 ± 0.01{per thousand}, respectively. The difference of Mfs-hprt between workers and controls was very significant (P < 0.01).

The results of TCR gene mutation assay in 21 workers and 21 controls
The results of Mf-TCR in workers and controls are exhibited in Table III. The ranges of Mf-TCR of workers and controls were 3.53–13.34 x 10–4 and 0.82–3.37 x 10–4, respectively. The average Mf-TCR of workers was 6.87 ± 0.52 x 10–4, which was significantly higher than that (1.67 ± 0.14 x 10–4) of controls (P < 0.01).

The correlation between six parameters and exposure years
The correlation between six parameters and exposure years is shown in Table IV. It was discovered that there was only a good correlation between exposure years and micronucleus formation (MCR and MNR) in exposed subjects; the correlation coefficients were 0.522 and 0.576, respectively. However, there was no good correlation between exposure years and other parameters (MTL, MTM, Mf-TCR and Mf-hprt).


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  		Table IV.. The correlation coefficients between six parameters and exposure year

 
The difference of six parameters between males and females among the 21 workers
The mutant frequencies of the TCR gene of the exposed male subjects and female subjects were 7.83 ± 0.78 and 5.81 ± 0.53 x 10–4, respectively (Table V). Mf-TCR of the exposed male subjects was significantly higher than that of the exposed female subjects (P < 0.01). The difference of other parameters between the exposed male subjects and the exposed female subjects was not significant (P > 0.05).


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  		Table V.. The difference of six parameters between males and females in 21 workers

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Conclusion
 References
 
The increasing MN frequency induced by MTX
The results of the study reported by Shahin et al. (19Go) clearly showed that MTX is a genotoxic drug. This was evidenced by the increased incidence of structural aberrations, particularly gaps, as well as numerical CAs in MTX-treated rats and rheumatoid arthritis cases. In the study of Shahin, the frequency of micronuclei was significantly increased in bone marrow cells as well as in peripheral blood cells of MTX-treated rats and rheumatoid arthritis patients, respectively. Moreover, several in vivo studies have indicated that multiple doses of MTX induced more micronuclei when compared to a single treatment in male mice. The MTX-induced micronuclei formation might be explained by the intracellular accumulation of the drug resulting in a continuous inhibition of deoxyribonucleotide triphosphate (dNTPs) synthesis, subsequently causing genetic lesions due to the inhibition of DNA repair. However, because insufficient dNTPs remain, the genetic lesions induced by MTX are expressed by micronuclei (20Go,30Go,31Go).

In the in vitro experiments performed by Keshava et al. (22Go), the results indicated that MTX alone induced a concentration-related increase in percentage MNBNs and percentage aberrant cells (Abs). There was a decrease in NDI with increase in MTX concentration. NDI reduction can be explained by the fact that the mechanism of MTX-induced genotoxicity is via inhibition of thymidylate, purines and glycine synthesis due to its inhibition of dihydrofolate reductase in vivo (21Go,22Go).

In the present investigation, similar results were observed in workers occupationally exposed to MTX. The results showed that the frequencies of MNBN and micronuclei (MN) in workers occupationally exposed to MTX increased significantly, compared with those in controls (P < 0.01). Meanwhile, there was a decrease in NDI of workers occupationally exposed to MTX. So the results of our investigation were in correspondence with the results of the above studies.

The DNA damage induced by MTX in the comet assay
Kopjar and Garaj-Vrhovac (5Go) used the alkaline comet assay to evaluate the genotoxicity towards peripheral lymphocytes of medical personnel regularly handling various antineoplastic drugs, including cyclophosphamide, vincristine, vinblastine, cis-platinum, 5-fluorouracil, bleomycin, MTX and adriamycin. Statistical differences in all three parameters were observed between the exposed and control groups. The present investigation is different from that study. In the above study, the subjects were occupationally exposed to many antineoplasic drugs, including drugs with known carcinogenic effects. In the present investigation, the 21 workers from a pharmaceutical plant were only exposed to MTX for which evidence for carcinogenicity in humans and animals is considered inadequate by IARC. The results of our study indicated that the MTL of exposed workers was significantly higher than that of controls in comet assay (P < 0.01), but there was no significant difference for MTM between the exposed group and control group (P > 0.05). We suppose that the contrary results may be due to the different sensitivity of MTL index and MTM index. It is possible that the sensitivity of MTL is higher than that of MTM.

The increasing Mf-hprt gene and Mf-TCR gene induced by MTX
The frequencies of somatic cell mutations at various housekeeping genes, including hypoxanthine-guanine phosphoribosyl-transferase (hprt), glycophorin A (GPA), and the T-cell receptor (TCR), have been employed to detect the genotoxic effects of tobacco use, dietary exposure, occupational exposure, and radiation (atomic bomb and accidental exposure), including chemotherapeutic exposure (24Go,32GoGoGoGoGo–37Go). So both of hprt assay and TCR assay have been used to detect the genetic hazard of workers occupationally exposed only to MTX in the present investigation. The results of hprt assay and TCR assay in our experiment indicated the significant difference between the exposed group and control group. The average Mf-hprt and Mf-TCR of workers occupationally exposed to MTX were 1.00 ± 0.02{per thousand} and 6.87 ± 0.52 x 10–4, respectively, which were significantly higher than those of controls (P < 0.01). According to the description by Kubota et al. (32Go) and Akiyama et al. (38Go), the mutant frequency of every one of TCR gene, hprt gene and GPA gene increased significantly with age. But it was found in the present experiment that Mfs-TCR did not increase with age in controls. The controls were divided into 3 sub-groups with age: A sub-group (seven persons, <30 years old); B sub-group (five persons, 30–39 years old) and C sub-group (nine persons, >40 years old). The average Mfs-TCR of A, B and C sub-groups were 1.94, 1.51 and 1.73 x 10–4, respectively. We supposed the reason may be that the sample was too small to express the relationship between Mfs-TCR and age.

The correlation between six parameters and exposure year
In this study, we discovered there was a good correlation only between exposure years and micronucleus formation in exposed subjects. It is difficult for us to explain the phenomenon. Perhaps, the phenomenon is associated with the end points. We propose that the enhancement of MCR and MNR expresses chromosomal damage. Chromosomal damage can pass from one cell cycle to the next cell cycle, last a long time and accumulate in damaged lymphocytes so that MCR and MNR increase with exposure years. However, this effect was not reflected at the level of DNA strand breaks. DNA strand breaks can be repaired by DNA repair pathways, so DNA strand breaks last for a shorter time. For example, our previous study showed that most of the DNA strand breaks of human lymphocytes induced by UVC were repaired by 240 min after UVC exposure (39Go).

The difference of six parameters between males and females in 21 workers
In the study, it is found that the mutant frequency of the TCR gene of the exposed male subjects was significantly higher than that of the exposed female subjects. Because Mf-TCR is associated with age (33Go), the average age was analyzed for male and female workers. The average age of 11 male workers is 32 years old; the average age of 10 female workers is 38 years old. As there was no significant difference between male average age and female average age (P = 0.165), the difference in mutation frequency may be due to a different sensitivity to MTX exposure for males and females.

The influence of protection measures on the genotoxic effects of MTX
Although 20 workers used protective measures (gloves and mask), six parameters of four tests increased significantly. It indicates that the protection measures do not prevent the genotoxic effects of MTX. A study in the Netherlands (2Go) showed a similar situation. The contamination of air and penetration through latex gloves were found for cyclophosphamide, 5-fluorouracil and MTX. The drugs were also found in urine samples of six technicians at higher levels than in the air, suggesting that inhalation is of minor importance for internal exposure compared with other routes, particularly dermal. Ündeger et al. (10Go) reported the permeability of polyethylene glove material to cyclophosphamide, 5-fluorouracil and MTX.


   Conclusion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
References
 
In this investigation, DNA damage, chromosomal damage and housekeeping gene mutation of 21 workers occupationally exposed to MTX were detected with the comet assay, MN test, hprt gene mutation assay and TCR gene mutation assay. The results showed that the cytogenetic markers of workers significantly increased when compared with controls.


   Acknowledgments
 
This research work was supported by International Cooperative Foundation of Science-Technique Bureau of Zhejiang Province (No. 012104).


   Notes
 
* To whom correspondence should be addressed. Zhejiang University, Medical College, Institute of Occupational and Environmental Health, Hangzhou 310006, Zhejiang, People's Republic of China. Tel: +86 571 87217188; Fax: +86 571 87217382; Email: he_jiliang{at}hotmail.com


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Conclusion
 References
 

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Received on May 2, 2005; revised and accepted on June 27, 2005


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