Joint effects of microwave and chromium trioxide on root tip cells of Vicia faba

Joint effects of microwave and chromium trioxide on root tip cells of Vicia faba first-author †E-mail:qianxiaowei@126.com †E-mail:qianxiaowei@126.com qianxiaowei@126.com School of Life and Environmental Science, Wenzhou University, Wenzhou 325003, China School of Life and Environmental Science, Wenzhou University, Wenzhou 325003, China The mutagenic effects of microwave and chromium trioxide (CrO 3 ) on Vicia faba root tip were studied. Micronucleus assay and chromosomal aberration assay were used to determine the mitotic index, the micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells induced by microwave and CrO 3 . The results showed that the micronucleus frequency decreased, and that the mitotic index and chromosomal aberration frequency showed linear dose responses to CrO 3 , in treatment of microwave for 5 s. In microwave of 25 s, the mitotic index decreased, the micronucleus frequency and chromosomal aberration frequency increased with increase of CrO 3 concentration. We concluded that microwave and CrO 3 had antagonistic effect on the mitotic index of Vicia faba root tip cells, but had synergetic effect on micronucleus frequency and chromosomal aberration frequency of Vicia faba root tip cells. Microwave Chromium trioxide (CrO 3 ) Antagonistic effect Synergetic effect INTRODUCTION 1 Electromagnetic radiation and microwave are human survival environmental factors. Their harmful effects on biology had been reported (Lacy-Hulbert et al., 1998 1998 ; Sher, 2000 2000 ; Forgacs et al., 2005 2005 ; Sripakdee et al., 2005 2005 ). Along with the popularization of network communication, broad-cast television, electric appliance, people accept more and more electromagnetic radiation, which has already become an environmental pollution problem, affecting humanity’s living conditions directly. Harm created by chromium alone has already been reported (Qian, 2004a 2004a ; Kim et al., 2004 2004 ; Garcia-Rodriguez et al., 2001 2001 ). But the joint effects of two factors have not been reported yet. This work studied the effects of microwave in different treatment time and chromium trioxide in different concentration on the mitotic index, the micronucleus frequency, and chromosomal aberration frequency of Vicia fabe root tip cells. MATERIALS AND METHODS 1 Materials 2 Vicia faba L. (collected from Hesheng Town, Yongjia of Zhejiang Province), CrO 3 (produced by Xilong Chemical Plant, Guangdong, at 12.5 mg/L and 50.0 mg/L in distilled water, Batch number 20010216). Microwave oven used was an NN-S547WFS model, made in Shanghai Panasonic Microwave L. CN. in 1997. Methods 2 Full size Vicia faba seeds were chosen. Table 1 1 presents the experimental grouping and treatment. The excised root tips were fixed in Carnoy’s fluid (anhydrous alcohol:glacial acetic acid=3:1, V / V ) for 24 h, and kept in 70% alcohol in refrigerator at 4 °C. These samples were sectioned routinely and stained with modified carbol fuchsin. The cell mitotic index, micronucleus permillage and chromosomal aberration percentage were examined and counted microscopically on squashes, and the aberrant cells were microphotographed (Qian, 2004b 2004b ). Each treatment as described above was repeated at least thrice; analysis of variance used SPSS software (SPSS, 10.0 version). Group Number of root tip ( n ) Time in oven (s)+concentration of CrO 3 (mg/L) A (control) 10 0+0 B 10 5+0 C 10 25+0 D 10 0+12.5 E 10 0+50.0 F 10 5+12.5 G 10 5+50.0 H 10 25+12.5 I 10 25+50.0 RESULTS 1 Mitotic indexes of Vicia faba root tip cells as induced by microwave and CrO 3 2 Table 2 2 shows obvious antagonistic effects. The mitotic indexes of Groups B and C were less than that of control (Group A) ( P < 0.05, P < 0.001). The mitotic index decreased significantly prolongation of microwave treatment time increased from 5 s to 25 s. Compared with Group A, the mitotic indexes of Groups D and E increased remarkably ( P < 0.001) when CrO 3 concentration increased. The mitotic indexes of Groups F and G were not remarkably different ( P > 0.05), showing that the mitotic index did not significantly change with increase of CrO 3 concentration for 5 s of microwave treatment. In Groups H and I the mitotic index decreased significantly with increasing CrO 3 concentration for 25 s of microwave treatment ( P < 0.001). The other groups compared with Group A P < 0.05 P < 0.001 Group Number of root tip ( n ) 10 Mitotic index for each root tip ( x ) (%) Mitotic index ( x ݱ s )(%) A (control) 10 3.9 4.0 3.2 3.9 3.8 3.0 4.1 4.8 4.4 3.7 3.88±0.52 B 10 3.7 3.6 3.4 3.4 3.8 3.3 3.5 3.1 3.2 3.7 3.47±0.23 * * C 10 3.3 3.0 3.4 2.9 3.6 3.4 3.1 2.8 2.7 2.9 3.11±0.30 *** *** D 10 5.4 5.5 5.1 5.6 5.1 5.2 4.5 4.5 4.8 4.9 5.06±0.39 *** *** E 10 5.6 5.3 5.4 5.7 5.3 5.2 5.1 5.0 5.1 5.5 5.32±0.23 *** *** F 10 3.7 4.1 3.7 3.1 3.7 3.7 3.2 4.4 3.3 3.8 3.67±0.40 G 10 4.2 4.0 4.1 4.2 3.9 4.5 4.0 4.3 3.9 3.9 4.10±0.20 H 10 2.8 3.2 2.9 2.9 3.2 3.4 2.8 3.7 3.5 3.5 3.19±0.33 *** *** I 10 2.2 2.8 3.0 2.4 3.6 3.4 2.7 3.4 3.6 2.6 2.97±0.51 *** *** Micronucleus frequency of Vicia faba root tip cells as induced by microwave and CrO 3 2 Table 3 3 shows the micronucleus frequencies of Groups B, C, D, and E were all higher than that of Group A ( P < 0.001). The micronucleus frequencies increased significantly with prolongation of microwave treatment time. The result revealed that treatment time determines the value of the micronucleus frequency ( P < 0.001). The micronucleus frequency of Group F was remarkably high compared to Groups B and D ( P < 0.001), and that of Group G was remarkably high compared to Groups B and E ( P < 0.001). These show combinations of 5 s microwave and 12.5 mg/L CrO 3 and 5 s and 50.0 mg/L had synergetic effects. The micronucleus frequency of Group H was lower than that of Group C, but higher than that of Group D ( P < 0.001). The micronucleus frequency of Group I was remarkably higher that that of Groups C and E ( P < 0.001). Synergetic effects of 25 s microwave and 50.0 mg/L CrO 3 on micronucleus frequency could be observed. The other groups compared with Group A P < 0.001 Group Number of root tip ( n ) Number of interphase cell observed 5 Number of cell with micronucleus per root tip Micronucleus frequency ( x ݱ s )(‰) 2 A (control) 2 10 2 1000 2 3 1 2 2 2 2.20±0.79 3 3 1 2 3 2 B 2 10 2 1000 11 11 10 12 10 2 10.60±0.97 *** *** 10 11 12 10 9 2 C 2 10 2 1000 14 15 16 17 16 2 15.40±0.97 *** *** 15 16 15 16 14 2 D 2 10 2 1000 9 11 12 13 10 2 10.40±1.35 *** *** 9 9 10 11 10 2 E 2 10 2 1000 5 6 6 7 5 2 6.60±1.17 *** *** 8 6 7 8 8 2 F 2 10 2 1000 25 26 28 27 26 2 25.10±1.79 *** *** 22 25 23 24 25 2 G 2 10 2 1000 17 16 14 15 16 2 14.80±1.23 *** *** 14 14 13 15 14 2 H 2 10 2 1000 14 13 15 13 12 2 12.50±1.58 *** *** 14 11 12 11 10 2 I 2 10 2 1000 20 20 18 20 17 2 19.20±1.23 *** *** 21 20 19 18 19 Chromosomal aberration of Vicia faba root tip cells as induced by microwave and CrO 3 2 Table 4 4 shows that the chromosomal aberration frequencies of the 8 test groups were remarkably higher than that of the control group ( P < 0.001). The chromosomal aberration frequency increased gradually with microwave treatment time and CrO 3 concentration ( P < 0.001). The chromosomal aberration frequency of Group F was remarkably higher than that of Groups B and D ( P < 0.001), and that of Group G was remarkably higher than that of Groups B and E ( P < 0.001, P < 0.01). This shows that two factors of 12.5 mg/L and 50.0 mg/L, 5 s had synergetic effect. The chromosomal aberration frequency of Group H was slightly higher than that of Group C ( P > 0.05), but apparently higher than that of Group D ( P < 0.001). These show that the two factors of 25 s and 12.5 mg/L had synergetic effect. The chromosome aberration frequency of Group I was slightly higher than that of Groups C ( P < 0.01) and E ( P > 0.05). The other groups compared with Group A P < 0.001 Group Number of root tips ( n ) 10 Number of cells with chromosomal aberration Chromosomal aberration frequency ( x ݱ s )(%) A (control) 10 2 2 1 1 3 1 1 2 3 2 1.80±0.79 B 10 20 21 18 17 17 20 17 19 19 20 18.80±1.48 *** *** C 10 26 27 27 28 30 28 28 29 30 29 28.20±1.32 *** *** D 10 20 21 18 17 19 17 17 19 20 19 18.70±1.42 *** *** E 10 31 30 32 28 29 33 31 32 29 28 30.30±1.77 *** *** F 10 27 29 28 28 31 28 27 28 30 27 28.30±1.34 *** *** G 10 35 31 32 36 31 30 33 32 34 31 32.50±1.96 *** *** H 10 31 29 28 27 30 31 30 28 29 32 29.50±1.58 *** *** I 10 32 30 29 34 28 27 30 31 30 33 30.40±2.17 *** *** Mitoses of Vicia faba affected by microwave and CrO 3 2 Microscope examination found abnormal cell mitosis phenomenon in prophase, metaphase, anaphase, telophase of Vicia faba root tip meristem cells in the presence of microwave and CrO 3 . 1. Prophase: The main feature is the occurrence of single or double micronuclei (Figs. 1a, 1b 1a, 1b , 2c 2c , 3b 3b and 4b 4b ). In the last mitosis the chromosome fragment is ejected from the cell nucleus, and then uncoils in the cytoplasm and becomes the micronucleus, formed in the cytoplasm. 2. Metaphase: Aside from the micronucleus (Figs. 1g 1g , 2e 2e and 3f 3f ), there is a chromosome that remained and diverged and was formed by spindle fiber broken in the mitosis process (Figs. 1f 1f , 3c 3c , 3d 3d , 4c 4c and 2d 2d ). The chromosomes in groups on the equatorial planes are resulted from the abnormal activities of chromosomes (Figs. 1e 1e and 3e 3e ) and multi-polar phenomena in these kinds of cells will appear in anaphase, when there is also chromosome blending (Figs. 1c, 1d 1c, 1d , 4d 4d and 4e 4e ). 3. Anaphase: The abnormality has mainly four features. The first is the occurrence of chromosomal bridge (Figs. 2f, 2g 2f, 2g , 2h 2h and 4f 4f ), one of the main features of abnormal cell division and chromosomal aberration, results from the formation of dicentric chromosome and acentric fragment. The formation of chromosomal bridges is accompanied by the occurrence of the chromosomal fragment (Fig. 2h 2h ). Secondly, chromosomal lagging appears (Figs. 1h 1h , 3g 3g and 3h 3h ). Most chromosomes move to both poles normally, only a few chromosomes or chromosomal fragments remained between the two poles. This reflects that the moving speed of the chromosomes differs from the rest. Thirdly, multi-polar phenomena occurs. In the anaphase of a normal mitosis, the chromosomes move equally to the opposite poles with the traction of the spindle fibers. However abnormal multi-poles unequal distribution was observed in the Vicia faba root tip cells which had been treated with microwave and CrO 3 (Fig. 4g 4g ). Fourthly, chromosomes stick to each other (Fig. 4g 4g ). 4. Telophase: The abnormality mainly includes the micronucleus (Figs. 3l 3l and 4l 4l ), the chromosomal fragment (Figs. 1j 1j , 2i 2i , 2j 2j and 4k 4k ), the chromosomal single bridge (Figs. 1k 1k , 2i 2i and 4i 4i ), the chromosomal double bridges (Figs. 1j 1j , 4j 4j and 4k 4k ), the chromosome resident (Fig. 3i 3i ), and abnormal uncoiling (Figs. 1i, 1k 1i, 1k , 2k, 2l 2k, 2l , 3j and 3k 3j and 3k ). The telophase micronucleus is possibly formed in the last mitosis or in the present mitosis (Figs. 3l 3l and 4l 4l ). Some chromosomes uncoil unsynchronously. Most chromosomes have already uncoiled spirally and became the chromatin, chromosomes which have not uncoiled spirally still exist as chromosome. 5. Polyploid: The spindle fiber is broken in the mitosis process, so that daughter chromosomes cannot reach the two poles. The mitosis process is prevented, as a result, daughter cell does not form and becomes polyploid (Fig. 4h 4h ). 6. Syncytium: Abnormal multi-poles unequal distribution and damage or abnormality of individual chromosome lead to syncytium in anaphase (Figs. 1l 1l , 2b 2b , 3a 3a and 4a 4a ). DISCUSSION 1 CrO 3 alone has a turnover dose-effect on the micronucleus frequency, possibly because the highly concentrated solution causes the cell mitosis to pause so it fails to enter into the next cell cycle, and consequently has lower micronucleus frequency. This accords with the results of previous work (Qian, 2004a 2004a ). Hexavalent chromium causes DNA damage in animals and men (Knasmuller et al., 1998 1998 ) and also shows higher toxicity than its trivalent compounds since chromium ions in the +6 oxidation state easily cross biological membranes (Szelag et al., 2003 2003 ). The results obtained in the present study showed that antagonistic effect appeared between specific concentrations of CrO 3 and microwave exposure time in the mitotic index, micronucleus frequency, and chromosomal aberration frequency. Implying that microwave weakened the effect of chromium on cell cycle by prolonging the mitosis cycle. Moreover, microwave may be the cause of antagonistic effect by reducing chromium ions from the +6 to the +3 oxidation state, which is of potential value in combating chromium poisoning. Repetitive high power microwave can exert inhibitory influence on the process of DNA and RNA syntheses in tumor cells of P-815 mastocytoma (Litviakov et al., 2005 2005 ). It was reported that microwaves can preserve not only cell and nuclear shapes but also immunohistochemical antigenicity. However, their enzyme function was lost, which indicated cell dysfunction and death (Ozaki et al., 2003 2003 ). 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Assessment of efficacy of quercetin-5'-sulfonic acid sodium salt in the treatment of acute chromium poisoning: experimental studies 10.1631/jzus.2006.B0221 Project supported by Wenzhou Technology Bureau (No. S2002A015) and Wenzhou Normal College (No. 2003Z20), China 2005-10-31 2005-12-13 2006-02-27 2022-01-05 Copyright © 2006, Journal of Zhejiang University Science 2006 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 03 07