Environ Sci Pollut Res (2014) 21:3363–3370 DOI 10.1007/s11356-013-2282-9
RESEARCH ARTICLE
Rhodamine B induces long nucleoplasmic bridges and other nuclear anomalies in Allium cepa root tip cells Dehong Tan & Bing Bai & Donghua Jiang & Lin Shi & Shunchang Cheng & Dongbing Tao & Shujuan Ji
Received: 28 May 2013 / Accepted: 24 October 2013 / Published online: 15 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract The cytogenetic toxicity of rhodamine B on root tip cells of Allium cepa was investigated. A. cepa were cultured in water (negative control), 10 ppm methyl methanesulfonate (positive control), and three concentrations of rhodamine B (200, 100, and 50 ppm) for 7 days. Rhodamine B inhibited mitotic activity; increased nuclear anomalies, including micronuclei, nuclear buds, and bridged nuclei; and induced oxidative stress in A. cepa root tissues. Furthermore, a substantial amount of long nucleoplasmic bridges were entangled together, and some nuclei were simultaneously linked to several other nuclei and to nuclear buds with nucleoplasmic bridges in rhodamine B-treated cells. In conclusion, rhodamine B induced cytogenetic effects in A. cepa root tip cells, which suggests that the A. cepa root is an ideal model system for detecting cellular interactions. Keywords Rhodamine B . Allium cepa . Nucleoplasmic bridges . Oxidative stress . Nuclear bud . Genotoxicity
Introduction Rhodamine B (Food Red 15, CAS Registry No. 81-88-9), a xanthene dye that is soluble in water, methanol, and ethanol, is commonly used for dyeing textiles, paper, soap, leather, and drugs. Although rhodamine B is approved for use as a food dye in some countries (Tripathy et al. 1995), it is generally considered to be a toxic substance. Rhodamine B can be Responsible editor: Markus Hecker D. Tan : B. Bai : D. Jiang : L. Shi : S. Cheng : D. Tao : S. Ji (*) College of Food, Shenyang Agricultural University, No. 120 of Dongling Road, Shenhe District, Shenyang 110866, People’s Republic of China e-mail: [email protected]
harmful if acutely swallowed by humans or animals, causing acute irritation to the skin, eyes, and respiratory tract, and can induce phototoxic and photoallergic reactions (Rochat et al. 1978). Furthermore, the carcinogenicity, genotoxicity, and chronic toxicity of rhodamine B have been experimentally confirmed (Rajeev et al. 2007). Rhodamine B has known carcinogenic effects in rats, producing local sarcomas following subcutaneous injections (Tripathy et al. 1995). Its mutagenic properties have also been demonstrated by a Bacillus subtilis tee-assay (Kada et al. 1972), a Saccharomyces cerevisiae mitotic gene conversion assay (Ito and Kobayashi 1977), an Escherichia coli assay (Luck et al. 1963), and a Chinese hamster ovary cell assay (Au and Hsu 1979; Nestmann et al. 1979). However, in a Salmonella assay, rhodamine B showed non-mutagenic properties, both in the presence and absence of exogenous metabolic systems (Muzzall and Cook 1979). Because rhodamine B shows a high degree of persistence, it is commonly used as a systemic marker in a variety of animals. For example, after feeding coypu (Myocastor coypus) 0.5 g kg−1 of rhodamine B for 3 days, visible traces remained in the underfur for up to 225 days (Fry et al. 2010). Such persistence makes the elimination of rhodamine B difficult, thereby increasing its potential health hazards. The pervasive amount of rhodamine B that is generally present in the environment (Merouani et al. 2010), along with its use as a food dye, may pose potential risks to human health. Although abundant data are available concerning the toxicological effects of rhodamine B in animals and microorganisms, the genotoxicity in other systems, such as plant systems (Allium cepa), have not been investigated, which may provide additional toxicological information of rhodamine B. Higher plants are eukaryotes that undergo mitosis, meiosis, and mutation, and their chromosome structure is similar to that of man (Evandri et al. 2000). A. cepa root tip is an excellent genetic model for detecting DNA damage, chromosome aberrations,
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and disturbances in the mitotic cycle; it has several advantages over other systems, such as low cost, ease of handling, and requiring no exogenous metabolic system. Furthermore, A. cepa root tip cells are sizable and contain large nuclei, thus facilitating their use in such investigations (Leme and MarinMorales 2009). Therefore, to further elucidate its genotoxic role, the cytogenetic toxicity of rhodamine B was investigated in A. cepa root tip cells in the present study.
Materials and methods Exposure schedule Healthy bulbs of A. cepa (purchased from a local supermarket in Shenyang city, China) were divided into five groups, with ten bulbs in each group. The bulbs were cleaned in running tap water for 25 min before use and were half immersed in only tap water as a negative control, 10 ppm methyl methanesulfonate (MMS, Sigma-Aldrich, St. Louis, USA) as a positive control (Saxena et al. 2005), or three concentrations of rhodamine B (Sigma-Aldrich, St. Louis, USA) (200, 100, and 50 ppm). Tested concentrations of rhodamine B were based on results of a preliminary assay that determined the concentration resulting in 50 % growth inhibition (IC50, 231.9 ppm; data not shown) of A. cepa roots for 7 days. This assay provided a reasonable range in which to investigate both toxic and nontoxic concentrations of rhodamine B. The bulbs were maintained at 20±2 °C, under a 12/12-h dark/light cycle in an artificial climate incubator (SAIFE PRX-45013, China), with the test solutions renewed every 24 h to ensure proper root growth. After 7 days of treatment, five root tips from each test bulb were cut and immediately fixed in freshly prepared Carnoy’s fixative (acetic acid to ethanol ratio, 1:3) for the mitotic activity and nuclear aberration assays. The remaining root tissues were cut from the bulbs; half were immediately used in the H2O2 assay, while the others were frozen at −80 °C until use in lipid peroxidation assays. Mitotic activity and nuclear aberration evaluation Slides were prepared according to methods described in Saxena et al. (2005). Briefly, root tips were fixed with Carnoy’s fixative solution for 24 h at 4 °C. The fixed root tips were then thoroughly washed with tap water, hydrolyzed with 1 N HCl, and stained with hematoxylin solution (Beijing Dingguo Changsheng Biotechnology Co. Ltd, China). The slides were scored for mitotic activity and nuclear aberrations under ×1,000 magnification with a light microscope and a digital capture system (Olympus 4X-1, Japan). Mitotic activity The mitotic index (MI) was determined by counting the number of mitotic cells among the total amount
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of scored cells per root tip. The frequency of cells in prophase, anaphase, metaphase, and telophase were scored in approximately 3,000 cells from five separate tips for each treatment and control group and were expressed as the percentage of total cells. Nuclear aberrations The frequencies of nuclear aberrations, including micronuclei (MN), nuclear buds (NBUDs), and bridged nuclei (BN), were evaluated and expressed as counts per 1,000 cells. The following criteria for aberrations in A. cepa root tip cells were used. MN: (a) approximately 1/10 to 1/3 the diameter of the main nucleus; (b) on the same focal plane with the main nucleus; (c) chromatin structure similar to that of the main nuclei; (d) smooth, oval, or round shape; and (e) clearly separated from the main nucleus (Ünyayar et al. 2006). NBUDs: Same morphology as MN, but bound to the nucleus by a narrow stalk of nucleoplasmic material (nucleoplasmic bridge) (Çavaş 2008). Cases in which multiple NBUDs were attached to a single nucleus were recorded as one NBUD for data processing. BN: Binucleated cell with nucleoplasmic bridge between nuclei (Çavaş 2008).
Oxidative stress assay H2O2 production The intracellular H2O2 as representative of reactive oxygen species (ROS) content was determined according to the methods described by Chong et al. (2004). Five-hundred milligrams of fresh roots was homogenized at 4 °C in 5 ml 0.1 % trichloroacetic acid (TCA) and centrifuged at 12,000 rpm for 15 min. A 0.5-ml aliquot of the supernatant was added to a solution containing 0.5 ml 10 mM potassium phosphate buffer (pH 7.0) and 1 ml 1 M potassium iodide. The absorbance was recorded at 390 nm on a spectrophotometer (Thermo Genesys 10, USA), and the H2O2 content was calculated based on a standard curve using gradual H2O2 concentrations. Lipid peroxidation Lipid peroxidation was determined by measuring the amount of malonaldehyde (MDA) according to methods described by Ünyayar et al. (2006). Frozen roots (100 mg) were homogenized by the addition of 1 ml 5 % TCA solution and then were centrifuged at 12,000 rpm for 15 min. The supernatant, along with equal volumes of 0.5 % thiobarbituric acid (TBA) in 20 % TCA solution (freshly prepared), were incubated at 96 °C for 25 min and then centrifuged at 10,000 rpm for 5 min. The absorbance of the supernatant was measured at 532 nm in a spectrophotometer and corrected by subtracting that at 600 nm; 0.5 % TBA in 20 % TCA solution was used as the blank. MDA content was determined using an extinction coefficient of 155 mM−1 cm−1.
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Antioxidant enzyme assays Activities of ROS scavenging enzymes (superoxide dismutase (SOD), ascorbic acid peroxidase (APX), catalase (CAT), and glutathione peroxidase (GPX)) were investigated. SOD removes O2− by catalyzing its dismutation, one O2− being reduced to H2O2 and another oxidized to O2, providing the first line of defense against the toxic effects of elevated levels of ROS (Gill and Tuteja 2010). CAT dismutate H2O2 into H2O and O2, and is indispensable for ROS detoxification during stressed conditions (Garg and Manchanda 2009). APX is involved in scavenging of H2O2 in ascorbic acid (ASH)–glutathione (GSH) cycle and utilizes ASH as the electron donor and is thought to play the most essential role in scavenging ROS and protecting cells in higher plants, algae, euglena, and other organisms (Gill and Tuteja 2010). GPX is a large family of diverse isozymes that use GSH to reduce H2O2 and organic and lipid hydroperoxides and therefore help plant cells from oxidative stress (Noctor et al. 2002). Breifly, fresh roots (250 mg) were homogenized in 10 ml of chilled 10 mM phosphate buffer solution (pH 7.0) under icecold conditions and centrifuged at 4 °C for 30 min at 15, 000 rpm. Protein content in the homogenate was determined as per Lowry et al. (1951) using bovine serum albumin as a standard. Activities of SOD were assayed by measuring its capacity to inhibit photochemical reduction of nitroblue tetrazolium (Beauchamp and Fridovich 1971). APX activity was determined as the oxidation of ASH to dehydroascorbate by measuring decrease in absorbance at 290 nm (Nakano and Asada 1981). CAT activity was examined by measuring rate of disappearance of H2O2 at 240 nm (Cakmak and Marschner 1992). GPX activity was tested according to the method described by Drotar et al. (1985).
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respectively) decreased significantly (p ≤0.05) compared with that of the negative control group (7.6±1.1 %), while that of the 50 ppm rhodamine B treatment group (7.3±0.9 %) was only slightly, and not significantly, lower than that of the negative control group. The MI of the positive control group (10 ppm MMS; 5.3±0.8 %) was also significantly reduced compared with that of the negative control group (Fig. 1). Nuclear aberration Overall, rhodamine B induced various nuclear aberrations in A. cepa root cells. In particular, a substantial amount of NBUDs and BN were observed, which were linked by short or long nucleoplasmic bridges (Figs. 2 and 3). NBUDs could be seen in interphase (Fig. 2b, c, f) and prophase of cells (Fig. 2d). Some nuclei were attached with multiple NBUDs, and several NBUDs possessed similar shapes and were neatly arranged (Fig. 2e, f). Other nuclear aberrations were also evident, such as MN (Fig. 2i) and chromosome breakages (Fig. 2h). Nucleoplasmic bridges between BN or between nuclei and NBUDs were clearly present. Some of these bridges were short, as shown in Fig. 2, which were located inside one cell, between two closely adjacent cells, or between cells and buds. These types of bridges are commonly observed in A. cepa root cells (Ghosh et al. 2010; Leme et al. 2008). Furthermore, many long nucleoplasmic bridges were observed in rhodamine B-treated A. cepa root cells. In some cases, these bridges were several times longer than the Upper
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Results Mitosis inhibition Rhodamine B induced a concentration-dependent reduction of root mitotic activity. The MI of both the 100 and 200 ppm rhodamine B treatment groups (5.8±0.4 % and 3.9±0.6 %,
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Fig. 1 Effect of rhodamine B on the mitotic index of root meristem cells of A. cepa. Upper panel microphotographs of representative normal mitotic A. cepa root tip cells. a Prophase cell. b Metaphase cell. c Anaphase cell. d Telophase cell. Scale bar 10 μm. Lower panel mitotic index of root meristem cells of A. cepa exposed to different concentrations of rhodamine B for 7 days. Data represent mean±SD of three independent experiments. *p ≤0.05, compared with negative control
3366 Fig. 2 Abnormal nuclei in A. cepa root cells induced by rhodamine B. a Bridged nuclei. b, c Nuclear bud in an interphase cell. d Nuclear bud in a prophase cell. e Double nuclear buds in a prophase cell. f Multiple-nuclear buds. g Nuclear bud with broken connection to nuclei in a metaphase cell. h Chromosome break in a metaphase cell. i Micronucleus. Scale bar 10 μm
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diameter of the nucleus (Fig. 3). Many of the long nucleoplasmic bridges formed bundles, and numerous bridges chaotically mixed to the web in some micro-areas (where data were excluded from processing due to the difficulty in distinguishing specific nuclear anomalies clearly) (Fig. 3d). For some nuclei, several long bridges formed links with other nuclei and with NBUDs (Fig. 2b). To our knowledge, this phenomenon has not been previously reported. The frequencies of NBUDs and BN were greatly and significantly enhanced, relative to those of the negative control (p ≤0.05), in a concentration-dependent manner within the tested range of rhodamine B concentration. In the 100 and 200 ppm rhodamine B groups, these frequencies even surpassed those of the positive control (MMS) group. The MN and chromosome breakage frequencies in the rhodamine B and positive control groups were all significantly higher than those in the negative control group (p ≤0.05) (Fig. 4).
Oxidative stress Levels of ROS (H2O2) and product of lipid peroxidation (MDA) (Fig. 5) increased in the rhodamine B groups in a
concentration-dependent manner; values obtained under all tested concentrations were significantly different from those of the negative control (p ≤0.05). The positive control (MMS) also induced high levels of H2O2 and MDA production. In general, activities of SOD, APX, CAT, and GPX showed a significant increase in all rhodamine B groups, compared with the negative control (Fig. 6); however, no clear concentration–response relationships were observed for CAT and GPX. The positive control also exhibited a significant increase in all enzyme activities.
Discussion Nuclear aberration assays are tools that have been extensively applied for detecting such genotoxic effects (Ali et al. 2009). The MN test is the most frequently applied method for nuclear aberration assays and has played an important role in providing early warnings of potential genotoxic threats (Tlili et al. 2010). MN result from chromosome fragments or whole chromosomes that either lag at cell division due to damage to the centromere or defect during the cell cleavage stage of
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Fig. 3 Long nucleoplasmic bridges in A. cepa root cells induced by rhodamine B. a Long nucleoplasmic bridge between the main nucleus and the bud. b Several long nucleoplasmic bridges string some nuclei and buds together in a series. c Multiple long nucleoplasmic bridges forming a bundle. d Numerous long nucleoplasmic bridges chaotically mixed to the web. 1 nucleus, 2 NBUD, arrows nucleoplasmic bridge. Scale bar 10 μm
the cell cycle, i.e., cytokinesis (Baršienė et al. 2006). In tissues with actively dividing cells, the frequency of MN reflects the action of clastogenic or aneugenic compounds (Baršienė et al. 2006). Various studies have found that A. cepa root tip cells have a high incidence of MN after exposure to different toxicants, such as pesticides, heavy metals, and their complex mixtures (Leme and Marin-Morales 2009). The results of the Fig. 4 The aberrant nuclei of A. cepa root tip cells induced by different concentrations of rhodamine B for 7 days. a Frequency of micronuclei. b Frequency of chromosome breakages. c Frequency of bridged nuclei. d Frequency of nuclear buds. Data represent mean±SD of three independent experiments. *p ≤0.05, compared with negative control. #p ≤0.05, compared with positive control
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present study confirmed those of previous reports, as rhodamine B induced an increase in the MN frequency, which indicates that rhodamine B possesses genotoxicity in A. cepa root tip cells. Along with MN, BN and NBUDs have received increasing research attention in the last 20 years and are generally regarded as biomarkers of genotoxic events (Fenech et al. 2011). BN stem from dicentric chromosomes, which may occur due to the misrepair of DNA breaks or telomere end fusions, or as a result of the defective separation of sister chromatids at anaphase due to decatenation failure. Rhodamine B was previously shown to induce BN in the broad bean, Vicia faba (Nestmann et al. 1979), which is consistent with results of the present study. The presence of NBUDs indicates the loss of amplified DNA, DNA repair complexes, and, possibly, excess chromosome accumulation from aneuploid cells (Fenech et al. 2011). One hypothesis to explain how genes may be amplified is the breakage–fusion–bridge (BFB) cycle model, which was originally proposed by B. McClintock (Shimizu et al. 2005). According to this model, BFB cycles are initiated by the formation of anaphase nucleoplasmic bridges, and the formation of BN, NBUDs, and MN occurs as a consequence of the cycle. Briefly, nucleoplasmic bridges between BN usually break unevenly, which results in one of the daughter cells receiving extra copies of genes while the other loses genes. Since such broken chromosomes lack telomere sequences at the broken end, they are prone to fuse with their replicas after DNA synthesis, which promotes the BFB cycle into the next rounds of cell division, resulting in further amplification of genes adjacent to the break or fusion point. Eventually, the
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Fig. 5 Rhodamine B-induced changes of H2O2 (a) and MDA (b) level in A. cepa roots. Data represent mean±SD of three independent experiments. *p ≤ 0.05, compared with negative control. #p ≤0.05, compared with positive control
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Fig. 6 Rhodamine B-induced relative changes of antioxidant enzyme activities in A. cepa roots. a SOD. b CAT. c APX. d GPX. Data represent mean±SD of three independent experiments. *p ≤0.05, compared with negative control
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elastic and therefore difficult to break. Second, although many NBUDs were observed, fewer MN (approximately 1/10 the number of NBUDs) were present in rhodamine B-exposed cells, suggesting that NBUDs might not always become MN. Third, multiple NBUDs found attached to the same nucleus possessed similar shapes and were neatly arranged, hinting that these NBUDs might not have originated from the broken nucleoplasmic bridges between BN. If they had originated from different broken bridges, the residue bridges would not show such equal lengths and neat arrangements, whereas if they stemmed from the same bridge, they might contain enough DNA chains or chromatin to be stable and difficult to break. Therefore, confirming the actual mechanisms underlying the results of the present study warrants further exploration. Interestingly, long nucleoplasmic bridges were observed to be chaotically mixed, and some nuclei were linked to several other nuclei and to NBUDs in A. cepa root tip cells. This
amplified genes are looped out of the aberrant chromosome by recombination, ultimately forming minute chromosomes (NBUDs), which can be eliminated by budding. If the connection linking the bud with the nucleus breaks before being excluded from the cell, the bud becomes MN (Shimizu et al. 1998, 2000). The above model has been used to explain the generation of a variety of gene amplifications or genomic instabilities related to cancer (Shimizu et al. 2005). The present finding that rhodamine B significantly induces NBUDs, as well as MN and BN, in tip cells of A. cepa indicates that rhodamine B causes genomic instability, which confirms its status as a carcinogen (Tripathy et al. 1995; Xie et al. 2010). Nevertheless, some results of the present study are not easily explained by the BFB theory. First, rhodamine B treatments increased the number of long nucleoplasmic bridges, which linked nuclei not only to other nuclei but also to NBUDs in some cases. The presence of so many long nucleoplasmic bridges might suggest that these bridges are very
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result reflects the efficiency of plant roots as genotoxicity model systems, since the root tissue remained relatively intact in the squash smear, making it easy to view relationships and interactions among cells. Bioaccumulation of toxic substances triggers redox reactions, generating ROS such as H2O2, superoxide anions (O2−), and hydroxyl radicals (HO−) (Sayeed et al. 2003), which induce a cascade of reactions that can be highly damaging to cells. ROS generation initiates the conversion of polyunsaturated fatty acids to lipid peroxides that are toxic at high concentrations; however, some also have important signaling functions affecting cell proliferation, apoptosis, and differentiation (Sawicki et al. 2003). To detoxify ROS and prevent cellular damage, plant cells and organelles like mitochondria and peroxisomes employ antioxidant defense systems (Gill and Tuteja 2010). The so-called oxidative stress reflects an imbalance between the systemic manifestation of ROS and the ability of biological systems to readily detoxify or repair the resulting damage (Lushchak 2011). The present study revealed a rhodamine B-induced increase of ROS (represented by H2O2) content and the degree of lipid peroxidation (represented by MDA—the final product of lipid peroxidation), indicating that rhodamine B induced oxidative damage in A. cepa. These results are consistent with reports of various toxicants in plants (Kumar et al. 2011; Pašková et al. 2006; Singh et al. 2009; Souguir et al. 2011). The antioxidant defense systems are enzymatic and nonenzymatic components; the former includes SOD, APX, GPX, and CAT, and the latter includes ASH, GSH, αtocopherol, carotenoids, and flavonoids. A great deal of research has established that the induction of the cellular antioxidant machinery is important for protection against various stresses (Gill and Tuteja 2010). The present study investigated the influence of rhodamine B on antioxidant enzymes of A. cepa roots. The results showed that rhodamine B induced antioxidant enzymes of SOD, CAT, APX, and GPX, which are consistent with the previous discussion that the induction of the cellular antioxidant machinery is important for protection against stresses, and further confirmed that rhodamine B induces ROS. The results showed unclear concentration–response relationship for some of these enzymes (CAT and GPX), which may be explained by excessive ROS causing oxidation of the enzymes themselves and inhibition of their activities. Overproduction of ROS is the direct cause of genotoxicity events. ROS can modify DNA in several ways, including base and sugar lesions, strand breaks, DNA–protein crosslinks, and base-free sites. In addition, lipid peroxidates are mutagenic molecules that also cause damage to DNA bases (Souguir et al. 2011). Such oxidative damage may partially explain the fact that various nuclear aberrants were observed in rhodamine B-exposed A. cepa. MI is a reliable parameter for determining the presence of cytotoxic compounds and is a
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suitable metric for biomonitoring toxin levels (Andrade et al. 2010). The observed reduction of MI in rhodamine B-treated roots was most likely due to disturbances in the cell cycle, as well as to chromatin dysfunction caused by the overproduction of ROS and lipid peroxides.
Conclusion In conclusion, rhodamine B induced oxidative stress, mitotic inhibition, as well as various nuclear aberrant patterns in A. cepa roots. In particular, several long nucleoplasmic bridges were observed in rhodamine B-treated tip cells of A. cepa, which have not been reported previously. Oxidative stress may be a partial mechanism contributing to these toxic phenomena. However, determining the actual toxicological mechanism(s) responsible, particularly for the long nucleoplasmic bridges, needs further investigation. Acknowledgments Financial support from the grants of the National Natural Science Foundation of China (31101220) and State Science and Technology Support Program (2012BAD38B07) is gratefully acknowledged. Conflict of interest The authors declare that there are no conflicts of interest.
References Ali D, Nagpure N, Kumar S, Kumar R, Kushwaha B, Lakra W (2009) Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Food Chem Toxicol 47: 650–656 Andrade L, Davide L, Gedraite L (2010) The effect of cyanide compounds, fluorides, aluminum, and inorganic oxides present in spent pot liner on germination and root tip cells of Lactuca sativa. Ecotoxicol Environ Saf 73:626–631 Au W, Hsu T (1979) Studies on the clastogenic effects of biologic stains and dyes. Environ Mutagen 1:27–35 Baršienė J, Dedonytė V, Rybakovas A, Andreikėnaitė L, Andersen OK (2006) Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil. Aquat Toxicol 78:S99–S104 Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44: 276–287 Cakmak I, Marschner H (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol 98: 1222–1227 Çavaş T (2008) In vivo genotoxicity of mercury chloride and lead acetate: Micronucleus test on acridine orange stained fish cells. Food Chem Toxicol 46:352–358 Chong TM, Abdullah MA, Fadzillah N, Lai OM, Lajis N (2004) Anthraquinones production, hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from
3370 intermediary and production medium strategies. Plant Cell Rep 22: 951–958 Classics Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275 Drotar A, Phelps P, Fall R (1985) Evidence for glutathione peroxidase activities in cultured plant cells. Plant Sci 42:35–40 Evandri MG, Tucci P, Bolle P (2000) Toxicological evaluation of commercial mineral water bottled in polyethylene terephthalate: a cytogenetic approach with Allium cepa . Food Addit Contam 17:1037–1045 Fenech M, Kirsch-Volders M, Natarajan A, Surralles J, Crott J, Parry J, Norppa H, Eastmond D, Tucker J, Thomas P (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26: 125–132 Fry TL, Atwood TC, Dunbar MR (2010) Evaluation of rhodamine B as a biomarker for raccoons. Hum Wildl Interact 4:275–282 Garg N, Manchanda G (2009) ROS generation in plants: boon or bane? Plant Biosyst 143:81–96 Ghosh M, Paul J, Sinha S, Mukherjee A (2010) Comparative evaluation of promutagens o-PDA, m-PDA and MH for genotoxic response in root cells of Allium cepa L. Nucleus 53:45–50 Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930 Ito T, Kobayashi K (1977) A survey of in vivo photodynamic activity of xanthenes, thiazines, and acridines in yeast cells. Photochem Photobiol 26:581–587 Kada T, Tutikawa K, Sadaie Y (1972) In vitro and host-mediated “recassay” procedures for screening chemical mutagens; and phloxine, a mutagenic red dye detected. Mutat Res-Fund Mol M 16:165–174 Kumar M, Trivedi N, Reddy C, Jha B (2011) Toxic effects of imidazolium ionic liquids on the green seaweed Ulva lactuca : oxidative stress and DNA damage. Chem Res Toxicol 24: 1882–1890 Leme DM, Marin-Morales MA (2009) Allium cepa test in environmental monitoring: a review on its application. Mutat Res Rev Mutat 682: 71–81 Leme DM, DdFd A, Marin-Morales MA (2008) Action mechanisms of petroleum hydrocarbons present in waters impacted by an oil spill on the genetic material of Allium cepa root cells. Aquat Toxicol 88: 214–219 Luck H, Wallnofer P, Bach H (1963) Food additives and mutagenic effects. 7th Report. Investigations of some xanthene dyes for mutagenic effects on Escherichia coli . Pathol Microbiol 26: 206–224 Lushchak VI (2011) Environmentally induced oxidative stress in aquatic animals. Aquat Toxicol 101:13–30 Merouani S, Hamdaoui O, Saoudi F, Chiha M (2010) Sonochemical degradation of rhodamine B in aqueous phase: effects of additives. Chem Eng J 158:550–557 Muzzall JM, Cook WL (1979) Mutagenicity test of dyes used in cosmetics with the Salmonella/mammalian-microsome test. Mutat Res Genet Toxicol 67:1–8 Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880
Environ Sci Pollut Res (2014) 21:3363–3370 Nestmann ER, Douglas GR, Matula TI, Grant CE, Kowbel DJ (1979) Mutagenic activity of rhodamine dyes and their impurities as detected by mutation induction in Salmonella and DNA damage in Chinese hamster ovary cells. Cancer Res 39:4412–4417 Noctor G, Gomez L, Vanacker H, Foyer CH (2002) Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling. J Exp Bot 53:1283–1304 Pašková V, Hilscherová K, Feldmannová M, Bláha L (2006) Toxic effects and oxidative stress in higher plants exposed to polycyclic aromatic hydrocarbons and their N‐heterocyclic derivatives. Environ Toxicol Chem 25:3238–3245 Rajeev J, Megha M, Shalini S, Alok M (2007) Removal of the hazardous dye rhodamine B through photocatalytic and adsorption treatments. J Environ Manage 85:956–964 Rochat J, Demenge P, Rerat JC (1978) Toxicologic study of a fluorescent tracer: rhodamine B. Toxicol Eur Res 1:23–26 Sawicki R, Singh SP, Mondal AK, Benes H, Zimniak P (2003) Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class. Biochem J 370:661–669 Saxena P, Chauhan L, Gupta S (2005) Cytogenetic effects of commercial formulation of cypermethrin in root meristem cells of Allium sativum: spectroscopic basis of chromosome damage. Toxicology 216:244–252 Sayeed I, Parvez S, Pandey S, Bin-Hafeez B, Haque R, Raisuddin S (2003) Oxidative stress biomarkers of exposure to deltamethrin in freshwater fish, Channa punctatus Bloch. Ecotoxicol Environ Saf 56:295–301 Shimizu N, Itoh N, Utiyama H, Wahl GM (1998) Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase. J Cell Biol 140:1307–1320 Shimizu N, Shimura T, Tanaka T (2000) Selective elimination of acentric double minutes from cancer cells through the extrusion of micronuclei. Mutat Res Fundam Mol Mech 448:81–90 Shimizu N, Shingaki K, Kaneko-Sasaguri Y, Hashizume T, Kanda T (2005) When, where and how the bridge breaks: anaphase bridge breakage plays a crucial role in gene amplification and HSR generation. Exp Cell Res 302:233–243 Singh HP, Kaur S, Batish DR, Sharma VP, Sharma N, Kohli RK (2009) Nitric oxide alleviates arsenic toxicity by reducing oxidative damage in the roots of Oryza sativa (rice). Nitric Oxide 20:289–297 Souguir D, Ferjani E, Ledoigt G, Goupil P (2011) Sequential effects of cadmium on genotoxicity and lipoperoxidation in Vicia faba roots. Ecotoxicology 20:329–336 Tlili S, Jebali J, Banni M, Haouas Z, Mlayah A, Helal AN, Boussetta H (2010) Multimarker approach analysis in common carp Cyprinus carpio sampled from three freshwater sites. Environ Monit Assess 168:285–298 Tripathy NK, Nabi MJ, Sahu GP, Kumar AA (1995) Genotoxicity testing of two red dyes in the somatic and germ line cells of Drosophila. Food Chem Toxicol 33:923–927 Ünyayar S, Çelik A, Çekiç FÖ, Gözel A (2006) Cadmium-induced genotoxicity, cytotoxicity and lipid peroxidation in Allium sativum and Vicia faba. Mutagenesis 21:77–81 Xie M, Jing L, Zhou J, Lin J, Fu H (2010) Synthesis of nanocrystalline anatase TiO 2 by one-pot two-phase separated hydrolysis– solvothermal processes and its high activity for photocatalytic degradation of rhodamine B. J Hazard Mater 176:139–145
RESEARCH ARTICLE
Rhodamine B induces long nucleoplasmic bridges and other nuclear anomalies in Allium cepa root tip cells Dehong Tan & Bing Bai & Donghua Jiang & Lin Shi & Shunchang Cheng & Dongbing Tao & Shujuan Ji
Received: 28 May 2013 / Accepted: 24 October 2013 / Published online: 15 November 2013 # Springer-Verlag Berlin Heidelberg 2013
Abstract The cytogenetic toxicity of rhodamine B on root tip cells of Allium cepa was investigated. A. cepa were cultured in water (negative control), 10 ppm methyl methanesulfonate (positive control), and three concentrations of rhodamine B (200, 100, and 50 ppm) for 7 days. Rhodamine B inhibited mitotic activity; increased nuclear anomalies, including micronuclei, nuclear buds, and bridged nuclei; and induced oxidative stress in A. cepa root tissues. Furthermore, a substantial amount of long nucleoplasmic bridges were entangled together, and some nuclei were simultaneously linked to several other nuclei and to nuclear buds with nucleoplasmic bridges in rhodamine B-treated cells. In conclusion, rhodamine B induced cytogenetic effects in A. cepa root tip cells, which suggests that the A. cepa root is an ideal model system for detecting cellular interactions. Keywords Rhodamine B . Allium cepa . Nucleoplasmic bridges . Oxidative stress . Nuclear bud . Genotoxicity
Introduction Rhodamine B (Food Red 15, CAS Registry No. 81-88-9), a xanthene dye that is soluble in water, methanol, and ethanol, is commonly used for dyeing textiles, paper, soap, leather, and drugs. Although rhodamine B is approved for use as a food dye in some countries (Tripathy et al. 1995), it is generally considered to be a toxic substance. Rhodamine B can be Responsible editor: Markus Hecker D. Tan : B. Bai : D. Jiang : L. Shi : S. Cheng : D. Tao : S. Ji (*) College of Food, Shenyang Agricultural University, No. 120 of Dongling Road, Shenhe District, Shenyang 110866, People’s Republic of China e-mail: [email protected]
harmful if acutely swallowed by humans or animals, causing acute irritation to the skin, eyes, and respiratory tract, and can induce phototoxic and photoallergic reactions (Rochat et al. 1978). Furthermore, the carcinogenicity, genotoxicity, and chronic toxicity of rhodamine B have been experimentally confirmed (Rajeev et al. 2007). Rhodamine B has known carcinogenic effects in rats, producing local sarcomas following subcutaneous injections (Tripathy et al. 1995). Its mutagenic properties have also been demonstrated by a Bacillus subtilis tee-assay (Kada et al. 1972), a Saccharomyces cerevisiae mitotic gene conversion assay (Ito and Kobayashi 1977), an Escherichia coli assay (Luck et al. 1963), and a Chinese hamster ovary cell assay (Au and Hsu 1979; Nestmann et al. 1979). However, in a Salmonella assay, rhodamine B showed non-mutagenic properties, both in the presence and absence of exogenous metabolic systems (Muzzall and Cook 1979). Because rhodamine B shows a high degree of persistence, it is commonly used as a systemic marker in a variety of animals. For example, after feeding coypu (Myocastor coypus) 0.5 g kg−1 of rhodamine B for 3 days, visible traces remained in the underfur for up to 225 days (Fry et al. 2010). Such persistence makes the elimination of rhodamine B difficult, thereby increasing its potential health hazards. The pervasive amount of rhodamine B that is generally present in the environment (Merouani et al. 2010), along with its use as a food dye, may pose potential risks to human health. Although abundant data are available concerning the toxicological effects of rhodamine B in animals and microorganisms, the genotoxicity in other systems, such as plant systems (Allium cepa), have not been investigated, which may provide additional toxicological information of rhodamine B. Higher plants are eukaryotes that undergo mitosis, meiosis, and mutation, and their chromosome structure is similar to that of man (Evandri et al. 2000). A. cepa root tip is an excellent genetic model for detecting DNA damage, chromosome aberrations,
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and disturbances in the mitotic cycle; it has several advantages over other systems, such as low cost, ease of handling, and requiring no exogenous metabolic system. Furthermore, A. cepa root tip cells are sizable and contain large nuclei, thus facilitating their use in such investigations (Leme and MarinMorales 2009). Therefore, to further elucidate its genotoxic role, the cytogenetic toxicity of rhodamine B was investigated in A. cepa root tip cells in the present study.
Materials and methods Exposure schedule Healthy bulbs of A. cepa (purchased from a local supermarket in Shenyang city, China) were divided into five groups, with ten bulbs in each group. The bulbs were cleaned in running tap water for 25 min before use and were half immersed in only tap water as a negative control, 10 ppm methyl methanesulfonate (MMS, Sigma-Aldrich, St. Louis, USA) as a positive control (Saxena et al. 2005), or three concentrations of rhodamine B (Sigma-Aldrich, St. Louis, USA) (200, 100, and 50 ppm). Tested concentrations of rhodamine B were based on results of a preliminary assay that determined the concentration resulting in 50 % growth inhibition (IC50, 231.9 ppm; data not shown) of A. cepa roots for 7 days. This assay provided a reasonable range in which to investigate both toxic and nontoxic concentrations of rhodamine B. The bulbs were maintained at 20±2 °C, under a 12/12-h dark/light cycle in an artificial climate incubator (SAIFE PRX-45013, China), with the test solutions renewed every 24 h to ensure proper root growth. After 7 days of treatment, five root tips from each test bulb were cut and immediately fixed in freshly prepared Carnoy’s fixative (acetic acid to ethanol ratio, 1:3) for the mitotic activity and nuclear aberration assays. The remaining root tissues were cut from the bulbs; half were immediately used in the H2O2 assay, while the others were frozen at −80 °C until use in lipid peroxidation assays. Mitotic activity and nuclear aberration evaluation Slides were prepared according to methods described in Saxena et al. (2005). Briefly, root tips were fixed with Carnoy’s fixative solution for 24 h at 4 °C. The fixed root tips were then thoroughly washed with tap water, hydrolyzed with 1 N HCl, and stained with hematoxylin solution (Beijing Dingguo Changsheng Biotechnology Co. Ltd, China). The slides were scored for mitotic activity and nuclear aberrations under ×1,000 magnification with a light microscope and a digital capture system (Olympus 4X-1, Japan). Mitotic activity The mitotic index (MI) was determined by counting the number of mitotic cells among the total amount
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of scored cells per root tip. The frequency of cells in prophase, anaphase, metaphase, and telophase were scored in approximately 3,000 cells from five separate tips for each treatment and control group and were expressed as the percentage of total cells. Nuclear aberrations The frequencies of nuclear aberrations, including micronuclei (MN), nuclear buds (NBUDs), and bridged nuclei (BN), were evaluated and expressed as counts per 1,000 cells. The following criteria for aberrations in A. cepa root tip cells were used. MN: (a) approximately 1/10 to 1/3 the diameter of the main nucleus; (b) on the same focal plane with the main nucleus; (c) chromatin structure similar to that of the main nuclei; (d) smooth, oval, or round shape; and (e) clearly separated from the main nucleus (Ünyayar et al. 2006). NBUDs: Same morphology as MN, but bound to the nucleus by a narrow stalk of nucleoplasmic material (nucleoplasmic bridge) (Çavaş 2008). Cases in which multiple NBUDs were attached to a single nucleus were recorded as one NBUD for data processing. BN: Binucleated cell with nucleoplasmic bridge between nuclei (Çavaş 2008).
Oxidative stress assay H2O2 production The intracellular H2O2 as representative of reactive oxygen species (ROS) content was determined according to the methods described by Chong et al. (2004). Five-hundred milligrams of fresh roots was homogenized at 4 °C in 5 ml 0.1 % trichloroacetic acid (TCA) and centrifuged at 12,000 rpm for 15 min. A 0.5-ml aliquot of the supernatant was added to a solution containing 0.5 ml 10 mM potassium phosphate buffer (pH 7.0) and 1 ml 1 M potassium iodide. The absorbance was recorded at 390 nm on a spectrophotometer (Thermo Genesys 10, USA), and the H2O2 content was calculated based on a standard curve using gradual H2O2 concentrations. Lipid peroxidation Lipid peroxidation was determined by measuring the amount of malonaldehyde (MDA) according to methods described by Ünyayar et al. (2006). Frozen roots (100 mg) were homogenized by the addition of 1 ml 5 % TCA solution and then were centrifuged at 12,000 rpm for 15 min. The supernatant, along with equal volumes of 0.5 % thiobarbituric acid (TBA) in 20 % TCA solution (freshly prepared), were incubated at 96 °C for 25 min and then centrifuged at 10,000 rpm for 5 min. The absorbance of the supernatant was measured at 532 nm in a spectrophotometer and corrected by subtracting that at 600 nm; 0.5 % TBA in 20 % TCA solution was used as the blank. MDA content was determined using an extinction coefficient of 155 mM−1 cm−1.
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Antioxidant enzyme assays Activities of ROS scavenging enzymes (superoxide dismutase (SOD), ascorbic acid peroxidase (APX), catalase (CAT), and glutathione peroxidase (GPX)) were investigated. SOD removes O2− by catalyzing its dismutation, one O2− being reduced to H2O2 and another oxidized to O2, providing the first line of defense against the toxic effects of elevated levels of ROS (Gill and Tuteja 2010). CAT dismutate H2O2 into H2O and O2, and is indispensable for ROS detoxification during stressed conditions (Garg and Manchanda 2009). APX is involved in scavenging of H2O2 in ascorbic acid (ASH)–glutathione (GSH) cycle and utilizes ASH as the electron donor and is thought to play the most essential role in scavenging ROS and protecting cells in higher plants, algae, euglena, and other organisms (Gill and Tuteja 2010). GPX is a large family of diverse isozymes that use GSH to reduce H2O2 and organic and lipid hydroperoxides and therefore help plant cells from oxidative stress (Noctor et al. 2002). Breifly, fresh roots (250 mg) were homogenized in 10 ml of chilled 10 mM phosphate buffer solution (pH 7.0) under icecold conditions and centrifuged at 4 °C for 30 min at 15, 000 rpm. Protein content in the homogenate was determined as per Lowry et al. (1951) using bovine serum albumin as a standard. Activities of SOD were assayed by measuring its capacity to inhibit photochemical reduction of nitroblue tetrazolium (Beauchamp and Fridovich 1971). APX activity was determined as the oxidation of ASH to dehydroascorbate by measuring decrease in absorbance at 290 nm (Nakano and Asada 1981). CAT activity was examined by measuring rate of disappearance of H2O2 at 240 nm (Cakmak and Marschner 1992). GPX activity was tested according to the method described by Drotar et al. (1985).
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respectively) decreased significantly (p ≤0.05) compared with that of the negative control group (7.6±1.1 %), while that of the 50 ppm rhodamine B treatment group (7.3±0.9 %) was only slightly, and not significantly, lower than that of the negative control group. The MI of the positive control group (10 ppm MMS; 5.3±0.8 %) was also significantly reduced compared with that of the negative control group (Fig. 1). Nuclear aberration Overall, rhodamine B induced various nuclear aberrations in A. cepa root cells. In particular, a substantial amount of NBUDs and BN were observed, which were linked by short or long nucleoplasmic bridges (Figs. 2 and 3). NBUDs could be seen in interphase (Fig. 2b, c, f) and prophase of cells (Fig. 2d). Some nuclei were attached with multiple NBUDs, and several NBUDs possessed similar shapes and were neatly arranged (Fig. 2e, f). Other nuclear aberrations were also evident, such as MN (Fig. 2i) and chromosome breakages (Fig. 2h). Nucleoplasmic bridges between BN or between nuclei and NBUDs were clearly present. Some of these bridges were short, as shown in Fig. 2, which were located inside one cell, between two closely adjacent cells, or between cells and buds. These types of bridges are commonly observed in A. cepa root cells (Ghosh et al. 2010; Leme et al. 2008). Furthermore, many long nucleoplasmic bridges were observed in rhodamine B-treated A. cepa root cells. In some cases, these bridges were several times longer than the Upper
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Results Mitosis inhibition Rhodamine B induced a concentration-dependent reduction of root mitotic activity. The MI of both the 100 and 200 ppm rhodamine B treatment groups (5.8±0.4 % and 3.9±0.6 %,
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Fig. 1 Effect of rhodamine B on the mitotic index of root meristem cells of A. cepa. Upper panel microphotographs of representative normal mitotic A. cepa root tip cells. a Prophase cell. b Metaphase cell. c Anaphase cell. d Telophase cell. Scale bar 10 μm. Lower panel mitotic index of root meristem cells of A. cepa exposed to different concentrations of rhodamine B for 7 days. Data represent mean±SD of three independent experiments. *p ≤0.05, compared with negative control
3366 Fig. 2 Abnormal nuclei in A. cepa root cells induced by rhodamine B. a Bridged nuclei. b, c Nuclear bud in an interphase cell. d Nuclear bud in a prophase cell. e Double nuclear buds in a prophase cell. f Multiple-nuclear buds. g Nuclear bud with broken connection to nuclei in a metaphase cell. h Chromosome break in a metaphase cell. i Micronucleus. Scale bar 10 μm
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diameter of the nucleus (Fig. 3). Many of the long nucleoplasmic bridges formed bundles, and numerous bridges chaotically mixed to the web in some micro-areas (where data were excluded from processing due to the difficulty in distinguishing specific nuclear anomalies clearly) (Fig. 3d). For some nuclei, several long bridges formed links with other nuclei and with NBUDs (Fig. 2b). To our knowledge, this phenomenon has not been previously reported. The frequencies of NBUDs and BN were greatly and significantly enhanced, relative to those of the negative control (p ≤0.05), in a concentration-dependent manner within the tested range of rhodamine B concentration. In the 100 and 200 ppm rhodamine B groups, these frequencies even surpassed those of the positive control (MMS) group. The MN and chromosome breakage frequencies in the rhodamine B and positive control groups were all significantly higher than those in the negative control group (p ≤0.05) (Fig. 4).
Oxidative stress Levels of ROS (H2O2) and product of lipid peroxidation (MDA) (Fig. 5) increased in the rhodamine B groups in a
concentration-dependent manner; values obtained under all tested concentrations were significantly different from those of the negative control (p ≤0.05). The positive control (MMS) also induced high levels of H2O2 and MDA production. In general, activities of SOD, APX, CAT, and GPX showed a significant increase in all rhodamine B groups, compared with the negative control (Fig. 6); however, no clear concentration–response relationships were observed for CAT and GPX. The positive control also exhibited a significant increase in all enzyme activities.
Discussion Nuclear aberration assays are tools that have been extensively applied for detecting such genotoxic effects (Ali et al. 2009). The MN test is the most frequently applied method for nuclear aberration assays and has played an important role in providing early warnings of potential genotoxic threats (Tlili et al. 2010). MN result from chromosome fragments or whole chromosomes that either lag at cell division due to damage to the centromere or defect during the cell cleavage stage of
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Fig. 3 Long nucleoplasmic bridges in A. cepa root cells induced by rhodamine B. a Long nucleoplasmic bridge between the main nucleus and the bud. b Several long nucleoplasmic bridges string some nuclei and buds together in a series. c Multiple long nucleoplasmic bridges forming a bundle. d Numerous long nucleoplasmic bridges chaotically mixed to the web. 1 nucleus, 2 NBUD, arrows nucleoplasmic bridge. Scale bar 10 μm
the cell cycle, i.e., cytokinesis (Baršienė et al. 2006). In tissues with actively dividing cells, the frequency of MN reflects the action of clastogenic or aneugenic compounds (Baršienė et al. 2006). Various studies have found that A. cepa root tip cells have a high incidence of MN after exposure to different toxicants, such as pesticides, heavy metals, and their complex mixtures (Leme and Marin-Morales 2009). The results of the Fig. 4 The aberrant nuclei of A. cepa root tip cells induced by different concentrations of rhodamine B for 7 days. a Frequency of micronuclei. b Frequency of chromosome breakages. c Frequency of bridged nuclei. d Frequency of nuclear buds. Data represent mean±SD of three independent experiments. *p ≤0.05, compared with negative control. #p ≤0.05, compared with positive control
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present study confirmed those of previous reports, as rhodamine B induced an increase in the MN frequency, which indicates that rhodamine B possesses genotoxicity in A. cepa root tip cells. Along with MN, BN and NBUDs have received increasing research attention in the last 20 years and are generally regarded as biomarkers of genotoxic events (Fenech et al. 2011). BN stem from dicentric chromosomes, which may occur due to the misrepair of DNA breaks or telomere end fusions, or as a result of the defective separation of sister chromatids at anaphase due to decatenation failure. Rhodamine B was previously shown to induce BN in the broad bean, Vicia faba (Nestmann et al. 1979), which is consistent with results of the present study. The presence of NBUDs indicates the loss of amplified DNA, DNA repair complexes, and, possibly, excess chromosome accumulation from aneuploid cells (Fenech et al. 2011). One hypothesis to explain how genes may be amplified is the breakage–fusion–bridge (BFB) cycle model, which was originally proposed by B. McClintock (Shimizu et al. 2005). According to this model, BFB cycles are initiated by the formation of anaphase nucleoplasmic bridges, and the formation of BN, NBUDs, and MN occurs as a consequence of the cycle. Briefly, nucleoplasmic bridges between BN usually break unevenly, which results in one of the daughter cells receiving extra copies of genes while the other loses genes. Since such broken chromosomes lack telomere sequences at the broken end, they are prone to fuse with their replicas after DNA synthesis, which promotes the BFB cycle into the next rounds of cell division, resulting in further amplification of genes adjacent to the break or fusion point. Eventually, the
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Fig. 5 Rhodamine B-induced changes of H2O2 (a) and MDA (b) level in A. cepa roots. Data represent mean±SD of three independent experiments. *p ≤ 0.05, compared with negative control. #p ≤0.05, compared with positive control
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elastic and therefore difficult to break. Second, although many NBUDs were observed, fewer MN (approximately 1/10 the number of NBUDs) were present in rhodamine B-exposed cells, suggesting that NBUDs might not always become MN. Third, multiple NBUDs found attached to the same nucleus possessed similar shapes and were neatly arranged, hinting that these NBUDs might not have originated from the broken nucleoplasmic bridges between BN. If they had originated from different broken bridges, the residue bridges would not show such equal lengths and neat arrangements, whereas if they stemmed from the same bridge, they might contain enough DNA chains or chromatin to be stable and difficult to break. Therefore, confirming the actual mechanisms underlying the results of the present study warrants further exploration. Interestingly, long nucleoplasmic bridges were observed to be chaotically mixed, and some nuclei were linked to several other nuclei and to NBUDs in A. cepa root tip cells. This
amplified genes are looped out of the aberrant chromosome by recombination, ultimately forming minute chromosomes (NBUDs), which can be eliminated by budding. If the connection linking the bud with the nucleus breaks before being excluded from the cell, the bud becomes MN (Shimizu et al. 1998, 2000). The above model has been used to explain the generation of a variety of gene amplifications or genomic instabilities related to cancer (Shimizu et al. 2005). The present finding that rhodamine B significantly induces NBUDs, as well as MN and BN, in tip cells of A. cepa indicates that rhodamine B causes genomic instability, which confirms its status as a carcinogen (Tripathy et al. 1995; Xie et al. 2010). Nevertheless, some results of the present study are not easily explained by the BFB theory. First, rhodamine B treatments increased the number of long nucleoplasmic bridges, which linked nuclei not only to other nuclei but also to NBUDs in some cases. The presence of so many long nucleoplasmic bridges might suggest that these bridges are very
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result reflects the efficiency of plant roots as genotoxicity model systems, since the root tissue remained relatively intact in the squash smear, making it easy to view relationships and interactions among cells. Bioaccumulation of toxic substances triggers redox reactions, generating ROS such as H2O2, superoxide anions (O2−), and hydroxyl radicals (HO−) (Sayeed et al. 2003), which induce a cascade of reactions that can be highly damaging to cells. ROS generation initiates the conversion of polyunsaturated fatty acids to lipid peroxides that are toxic at high concentrations; however, some also have important signaling functions affecting cell proliferation, apoptosis, and differentiation (Sawicki et al. 2003). To detoxify ROS and prevent cellular damage, plant cells and organelles like mitochondria and peroxisomes employ antioxidant defense systems (Gill and Tuteja 2010). The so-called oxidative stress reflects an imbalance between the systemic manifestation of ROS and the ability of biological systems to readily detoxify or repair the resulting damage (Lushchak 2011). The present study revealed a rhodamine B-induced increase of ROS (represented by H2O2) content and the degree of lipid peroxidation (represented by MDA—the final product of lipid peroxidation), indicating that rhodamine B induced oxidative damage in A. cepa. These results are consistent with reports of various toxicants in plants (Kumar et al. 2011; Pašková et al. 2006; Singh et al. 2009; Souguir et al. 2011). The antioxidant defense systems are enzymatic and nonenzymatic components; the former includes SOD, APX, GPX, and CAT, and the latter includes ASH, GSH, αtocopherol, carotenoids, and flavonoids. A great deal of research has established that the induction of the cellular antioxidant machinery is important for protection against various stresses (Gill and Tuteja 2010). The present study investigated the influence of rhodamine B on antioxidant enzymes of A. cepa roots. The results showed that rhodamine B induced antioxidant enzymes of SOD, CAT, APX, and GPX, which are consistent with the previous discussion that the induction of the cellular antioxidant machinery is important for protection against stresses, and further confirmed that rhodamine B induces ROS. The results showed unclear concentration–response relationship for some of these enzymes (CAT and GPX), which may be explained by excessive ROS causing oxidation of the enzymes themselves and inhibition of their activities. Overproduction of ROS is the direct cause of genotoxicity events. ROS can modify DNA in several ways, including base and sugar lesions, strand breaks, DNA–protein crosslinks, and base-free sites. In addition, lipid peroxidates are mutagenic molecules that also cause damage to DNA bases (Souguir et al. 2011). Such oxidative damage may partially explain the fact that various nuclear aberrants were observed in rhodamine B-exposed A. cepa. MI is a reliable parameter for determining the presence of cytotoxic compounds and is a
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suitable metric for biomonitoring toxin levels (Andrade et al. 2010). The observed reduction of MI in rhodamine B-treated roots was most likely due to disturbances in the cell cycle, as well as to chromatin dysfunction caused by the overproduction of ROS and lipid peroxides.
Conclusion In conclusion, rhodamine B induced oxidative stress, mitotic inhibition, as well as various nuclear aberrant patterns in A. cepa roots. In particular, several long nucleoplasmic bridges were observed in rhodamine B-treated tip cells of A. cepa, which have not been reported previously. Oxidative stress may be a partial mechanism contributing to these toxic phenomena. However, determining the actual toxicological mechanism(s) responsible, particularly for the long nucleoplasmic bridges, needs further investigation. Acknowledgments Financial support from the grants of the National Natural Science Foundation of China (31101220) and State Science and Technology Support Program (2012BAD38B07) is gratefully acknowledged. Conflict of interest The authors declare that there are no conflicts of interest.
References Ali D, Nagpure N, Kumar S, Kumar R, Kushwaha B, Lakra W (2009) Assessment of genotoxic and mutagenic effects of chlorpyrifos in freshwater fish Channa punctatus (Bloch) using micronucleus assay and alkaline single-cell gel electrophoresis. Food Chem Toxicol 47: 650–656 Andrade L, Davide L, Gedraite L (2010) The effect of cyanide compounds, fluorides, aluminum, and inorganic oxides present in spent pot liner on germination and root tip cells of Lactuca sativa. Ecotoxicol Environ Saf 73:626–631 Au W, Hsu T (1979) Studies on the clastogenic effects of biologic stains and dyes. Environ Mutagen 1:27–35 Baršienė J, Dedonytė V, Rybakovas A, Andreikėnaitė L, Andersen OK (2006) Investigation of micronuclei and other nuclear abnormalities in peripheral blood and kidney of marine fish treated with crude oil. Aquat Toxicol 78:S99–S104 Beauchamp C, Fridovich I (1971) Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44: 276–287 Cakmak I, Marschner H (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol 98: 1222–1227 Çavaş T (2008) In vivo genotoxicity of mercury chloride and lead acetate: Micronucleus test on acridine orange stained fish cells. Food Chem Toxicol 46:352–358 Chong TM, Abdullah MA, Fadzillah N, Lai OM, Lajis N (2004) Anthraquinones production, hydrogen peroxide level and antioxidant vitamins in Morinda elliptica cell suspension cultures from
3370 intermediary and production medium strategies. Plant Cell Rep 22: 951–958 Classics Lowry O, Rosebrough N, Farr A, Randall R (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275 Drotar A, Phelps P, Fall R (1985) Evidence for glutathione peroxidase activities in cultured plant cells. Plant Sci 42:35–40 Evandri MG, Tucci P, Bolle P (2000) Toxicological evaluation of commercial mineral water bottled in polyethylene terephthalate: a cytogenetic approach with Allium cepa . Food Addit Contam 17:1037–1045 Fenech M, Kirsch-Volders M, Natarajan A, Surralles J, Crott J, Parry J, Norppa H, Eastmond D, Tucker J, Thomas P (2011) Molecular mechanisms of micronucleus, nucleoplasmic bridge and nuclear bud formation in mammalian and human cells. Mutagenesis 26: 125–132 Fry TL, Atwood TC, Dunbar MR (2010) Evaluation of rhodamine B as a biomarker for raccoons. Hum Wildl Interact 4:275–282 Garg N, Manchanda G (2009) ROS generation in plants: boon or bane? Plant Biosyst 143:81–96 Ghosh M, Paul J, Sinha S, Mukherjee A (2010) Comparative evaluation of promutagens o-PDA, m-PDA and MH for genotoxic response in root cells of Allium cepa L. Nucleus 53:45–50 Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930 Ito T, Kobayashi K (1977) A survey of in vivo photodynamic activity of xanthenes, thiazines, and acridines in yeast cells. Photochem Photobiol 26:581–587 Kada T, Tutikawa K, Sadaie Y (1972) In vitro and host-mediated “recassay” procedures for screening chemical mutagens; and phloxine, a mutagenic red dye detected. Mutat Res-Fund Mol M 16:165–174 Kumar M, Trivedi N, Reddy C, Jha B (2011) Toxic effects of imidazolium ionic liquids on the green seaweed Ulva lactuca : oxidative stress and DNA damage. Chem Res Toxicol 24: 1882–1890 Leme DM, Marin-Morales MA (2009) Allium cepa test in environmental monitoring: a review on its application. Mutat Res Rev Mutat 682: 71–81 Leme DM, DdFd A, Marin-Morales MA (2008) Action mechanisms of petroleum hydrocarbons present in waters impacted by an oil spill on the genetic material of Allium cepa root cells. Aquat Toxicol 88: 214–219 Luck H, Wallnofer P, Bach H (1963) Food additives and mutagenic effects. 7th Report. Investigations of some xanthene dyes for mutagenic effects on Escherichia coli . Pathol Microbiol 26: 206–224 Lushchak VI (2011) Environmentally induced oxidative stress in aquatic animals. Aquat Toxicol 101:13–30 Merouani S, Hamdaoui O, Saoudi F, Chiha M (2010) Sonochemical degradation of rhodamine B in aqueous phase: effects of additives. Chem Eng J 158:550–557 Muzzall JM, Cook WL (1979) Mutagenicity test of dyes used in cosmetics with the Salmonella/mammalian-microsome test. Mutat Res Genet Toxicol 67:1–8 Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880
Environ Sci Pollut Res (2014) 21:3363–3370 Nestmann ER, Douglas GR, Matula TI, Grant CE, Kowbel DJ (1979) Mutagenic activity of rhodamine dyes and their impurities as detected by mutation induction in Salmonella and DNA damage in Chinese hamster ovary cells. Cancer Res 39:4412–4417 Noctor G, Gomez L, Vanacker H, Foyer CH (2002) Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling. J Exp Bot 53:1283–1304 Pašková V, Hilscherová K, Feldmannová M, Bláha L (2006) Toxic effects and oxidative stress in higher plants exposed to polycyclic aromatic hydrocarbons and their N‐heterocyclic derivatives. Environ Toxicol Chem 25:3238–3245 Rajeev J, Megha M, Shalini S, Alok M (2007) Removal of the hazardous dye rhodamine B through photocatalytic and adsorption treatments. J Environ Manage 85:956–964 Rochat J, Demenge P, Rerat JC (1978) Toxicologic study of a fluorescent tracer: rhodamine B. Toxicol Eur Res 1:23–26 Sawicki R, Singh SP, Mondal AK, Benes H, Zimniak P (2003) Cloning, expression and biochemical characterization of one Epsilon-class (GST-3) and ten Delta-class (GST-1) glutathione S-transferases from Drosophila melanogaster, and identification of additional nine members of the Epsilon class. Biochem J 370:661–669 Saxena P, Chauhan L, Gupta S (2005) Cytogenetic effects of commercial formulation of cypermethrin in root meristem cells of Allium sativum: spectroscopic basis of chromosome damage. Toxicology 216:244–252 Sayeed I, Parvez S, Pandey S, Bin-Hafeez B, Haque R, Raisuddin S (2003) Oxidative stress biomarkers of exposure to deltamethrin in freshwater fish, Channa punctatus Bloch. Ecotoxicol Environ Saf 56:295–301 Shimizu N, Itoh N, Utiyama H, Wahl GM (1998) Selective entrapment of extrachromosomally amplified DNA by nuclear budding and micronucleation during S phase. J Cell Biol 140:1307–1320 Shimizu N, Shimura T, Tanaka T (2000) Selective elimination of acentric double minutes from cancer cells through the extrusion of micronuclei. Mutat Res Fundam Mol Mech 448:81–90 Shimizu N, Shingaki K, Kaneko-Sasaguri Y, Hashizume T, Kanda T (2005) When, where and how the bridge breaks: anaphase bridge breakage plays a crucial role in gene amplification and HSR generation. Exp Cell Res 302:233–243 Singh HP, Kaur S, Batish DR, Sharma VP, Sharma N, Kohli RK (2009) Nitric oxide alleviates arsenic toxicity by reducing oxidative damage in the roots of Oryza sativa (rice). Nitric Oxide 20:289–297 Souguir D, Ferjani E, Ledoigt G, Goupil P (2011) Sequential effects of cadmium on genotoxicity and lipoperoxidation in Vicia faba roots. Ecotoxicology 20:329–336 Tlili S, Jebali J, Banni M, Haouas Z, Mlayah A, Helal AN, Boussetta H (2010) Multimarker approach analysis in common carp Cyprinus carpio sampled from three freshwater sites. Environ Monit Assess 168:285–298 Tripathy NK, Nabi MJ, Sahu GP, Kumar AA (1995) Genotoxicity testing of two red dyes in the somatic and germ line cells of Drosophila. Food Chem Toxicol 33:923–927 Ünyayar S, Çelik A, Çekiç FÖ, Gözel A (2006) Cadmium-induced genotoxicity, cytotoxicity and lipid peroxidation in Allium sativum and Vicia faba. Mutagenesis 21:77–81 Xie M, Jing L, Zhou J, Lin J, Fu H (2010) Synthesis of nanocrystalline anatase TiO 2 by one-pot two-phase separated hydrolysis– solvothermal processes and its high activity for photocatalytic degradation of rhodamine B. J Hazard Mater 176:139–145
