| 1.ObjectivesThe widespread utilization of chemotherapy in the treatment of cancer has led to anxiety about the possible hazards to staffs 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. In these studies, the used methods to detect cytogenetic damage include 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 varcinogenicity of MTX is considered inadequate by IARC, MTX can induce SCEs, CAs and MN. Vincristine(VCR) is an antineoplastic drug with the antimitotic action mode, also vincristine is one often anticancer agents which have been classified into group 1 (carcinogenic to humans) by IARC (1987, 1990). Because VCR is an spidle , VCR can impact mitosis and meiosis and induce the polyploidy, chromosomal aberrations and micronelei. Even VCR at lower dose has teratogenic effect. In order to determine whether the genetic damage appears in the workers only occupationally exposed to methotrexate or vincristine , in the present investigation three genetic endpoints, i.e. chromosomal damage ( micronucleus test), gene mutation (hprt gene mutation assay and TCR gene mutation assay) and DNA damage (comet assay) were utilized.2.Materials and methods 2.1 SamplesThe peripheral blood samples were collected from the workers occupationally exposed to MTX and VCR, and the cotrols matched with workers according to age, sex and smoking.The workers group exposed to MTX 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. There were no significant difference between workers and controls for gender, age and smoking habits. 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, testingand compounding. The exposure situation for workers is similar.The workers group exposed to VCR consisted of 6 males and 5 females from 36 to 52 years old (mean age, 43 years old), who were from a workshop producing vincrisitine. The average ages of male workers and female workers are 44 and 43 years old, respectively. The difference between male workers' average age and female workers' average age is not significant (P =0.733). The controls were matched with workers on the basis of age (from 36 to 55 years old, mean age, 43 years old), gender and smoking. There were no significant difference between workers and controls for gender, age and smoking habits. The area of the workplace is only about 300 m2 without good ventilating equipment, and 15 workers used protective measures (gloves and masks). The individual procedures include-.refining and synthesizeing.2.2 MethodsComet assay: Human lymphocytes were isolated and resuspended in PBS. Then cells were mixed with trypan blue solution and checked for viability. The following steps: slide preparation, alkaline lysis, DNA unwinding, electrophoresis and stain were performed. The comet lengths and moment were measured under a fluorescence microscope(OLYMPUS-BX51).The TCR gene mutation assay: Human lymphocytes were isolated and resuspended in PBS. Then cells were mixed with trypan blue solution and checked for viability and accounting cell number. The peripheral blood mononuclear cells were stained with fluorescein isothiocyanate (FITC)-labeled anti-CD4 MoAb and phycoerythrin (PE)-labeled anti-CD3 MoAb obtained from Becton Dickinson Immunocytometry System ,mix well, and incubate for 30 min on ice. TCR mutant CD4+T cells (CD3~CD4+) were analyzed by Coulter EPICS XL(Coulter Co., Hialeah, Florida, U.S.A.) with System II software. Mutant frequency of hprt gene (Mf-TCR) was calculated 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.Micronucleus test: The cultures were incubated at 37°C for 72 h. 4.5 ug/mlcytochalasin B was added to each culture at 28 h before harvesting cells. Then slides were prepared by cyto-centrifugation, fixed with methanol and stain. 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 were scored at 400>99%. The average MTL of workers was 1.30±0.06um, which was significantly higher than that (0.07±0.01um) of controls (pO.Ol). While the average MTM of 21 workers and 21 controls were 0.23±O.03 and 0.17±0.04, respectively. The difference between workers and controls for MTM was not significant (p>0.05).Micronucleus test: The mean MCRs of 21 workers and 21 controls were 8.05±0.75%o and 4.38±0.58 96o, respectively. The mean MCR of workers was significantly higher than that of controls (p<0.01). The average MNR of workers was 10.10±0.95 %o, which was significantly higher than that (5.48±0.82 %o) of controls (p<0.01). The average NDI of workers was 1.87±0.02, which was less than that (2.00±0.05) of controls (p<0.05).The hprt gene mutation assay and the TCR gene mutation assay: The ranges of Mf-hprt for workers and controls were 0.89-1.20 and 0.76-0.93%o, respectively. The average Mfs-hprt for workers and controls were 1.00±0.02%o and 0.86±0.01%o,respectively. The difference of Mfs-hprt between workers and controls was very significant (/K0.01). The ranges of Mf-TCR of workers and controls were 3.53-13.34X10"4 and 0.83-3.37x10"*, respectively. The average Mf-TCR of workers was 6.87±0.52xl0^, which was significantly higher than that (1.67±0.14xl0"4 )of controls (jXO.Ql).The correlation of six parameters for workers: We found that only there was a good correlation between MCR and MNR in micronucleus test, MTL and MTM in comet assay, and MTM and MCR(MNR) for different assays, the good correlation between 2 parameters for other different assays is not observed.The correlation between six parameters and exposure year: It was discovered that only there was 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 not any good correlation between exposure years and other parameters (MTL, MTM, Mf-TCR and Mf-hprt).3.2 The results of investigation for workers occupationally exposed to VCRComet assay: The cell viability was >99%. The average MTL of workers was 1.72±0.15um, which was significantly higher than that (0.71±0.01um) of controls (p<0.01). While the average MTM of 15 workers and 15 controls were 0.29±O..03 and 0.17±0.05, respectively. The difference between workers and controls for MTM was not significant (p>0.05).Micronucleus test: The mean MCRs of 15 workers and 15 controls werel3.67±1.56%o and 3.13±0.59%o, respectively. The mean MCR of workers was significantly higher than that of controls (p<0.01). The average MNR of workers was 17.80±1.88%o, which was significantly higher than that (3.73±0.80%o) of controls (p<0.01). The average NDI of workers was 1.94±0.05, which was less than that (1.99±0.04) of controls ,but The difference between workers and controls for NDI was not significant (p>0.05).The hprt gene mutation assay and the TCR gene mutation assay: The ranges ofMf-hprt for workers and controls were 0.89-1.14 %o and 0.78-0.94%o, respectively. The average Mfs-hprt for workers and controls were 1.03±0.02%o and 0.87±0.01%o, respectively. The difference of Mfs-hprt between workers and controls was very significant (p<0.01). The ranges of Mf-TCR of workers and controls were (1.06-5.08)xl0"4 and (0.84-2.12)xl0"4, respectively. The average Mf-TCR of workers was (2.52±0.34)xl0'4, which was significantly higher than that (l.SliO.l^xlO"4 of controls (p<0.05).The correlation of six parameters for workers: We found that only there was a good correlation between MCR and MNR in micronucleus test, MTL and MTM in comet assay, and MTM and TCR for different assays, the good correlation between 2 parameters for other different assays is not observed.The correlation between six parameters and exposure year: It was discovered that there was not any good correlation between exposure years and the parameters.4.ConclusionThe results showed that the cytogenetic markers of workers significantly increased when compared with controls.
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