SHORT COMMUNICATION Acridine orange/Ethidium bromide double ...

Allelopathy Journal 26 (2): 301-308 (2010) Table: -, Figs : 3

0971-4693/94 US $ 5.00 International Allelopathy Foundation 2010

SHORT COMMUNICATION Acridine orange/Ethidium bromide double staining test: A simple In-vitro assay to detect apoptosis induced by phenolic compounds in plant cells C. CINIGLIA1*, G. PINTO2, C. SANSONE1 and A. POLLIO2. 1 Department of Life Science, Second University of Naples, Via Vivaldi 43 Caserta 81100, Italy E. Mail: [email protected]

(Received in revised form: July 27, 2010)

ABSTRACT A wide range of genotoxicity assays are available to determine the DNA damage in plant cells caused by allelochemicals. However most of them, sometimes present some technical difficulties in interpretation of results. Acridine orange/ethidium bromide double staining assay is proposed as a rapid, inexpensive and easy-to-perform assay to investigate the apoptotic damage from phenols in plant cells. Our data support the validity of this assay and the hypothesis that phenols induce unrepairable severe genetic alterations due to apoptosis. The proapoptotic effects of Olive oil Mill wastewaters (OMWW, rich in polyphenols and catechol) were investigated on radish root cells. In 25% OMWW treated root cells, strong DNA fragmentations and hypersegmented nuclei were observed and increase in percentage of total apoptotic cells was registered during 24 h. The root cells treated with 12.5% OMWW showed the typical apoptotic hallmarks, but the percentage of total apoptotic cells decreased from 92% to 60% during 24 h, due to the significant decrease in early apoptotic cells and a concomitant increase in cell viability. The catechol at 10-4 to 10-3 M concentrations caused dose-dependent effect, nuclear damage increased during the 24 h and the root cells showed drastic chromatin disintegrations. Key words: Acridine orange, allelochemicals, apoptosis, catechol, ethidium bromide, olive mill wastewaters, phenols, Raphanus sativus

INTRODUCTION Few studies have addressed the problems of nuclear and DNA damages in plants cells exposed to allelochemical compounds. Recent studies have shown that the allelochemicals cause the genotoxic and cytotoxic effects. Ozmen and Tulay (11) determined the chromosomal and mitotic aberrations of Citrus spp. extracts in root meristems cells of Allium cepa L. Sousa et al. (13) also showed the allelopathic and genotoxic effects of Casearia sylvestris extracts on Allium cepa. Sunar et al. (14) tested the genotoxic effects of methanolic extracts of Verbascum speciosum on corn seeds using the RAPD (PCR-based random amplified polymorphic DNAs) technique. Correspondence author; 2Department of Biological Sciences, Section Plant Biology, University Federico II, Via Foria, 223, Naples, 80139, Italy

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The allelochemicals act directly or indirectly by causing the oxidative stress and inducing the subsequent necrosis or apoptosis (9). Little is known about apoptosis in plants, although recent studies have shown that some characteristics of apoptosis (disintegration and chromatin condensation) in plants are similar to animals (10,12). Morphological changes in nuclei structure (especially nuclear segmentation is an early and unequivocal hallmark of apoptosis) coupled with a loss of DNA integrity, which ultimately may result in cell death. Acridine orange/ethidium bromide (AO/EB) double staining test is fast and statistically significant fluorescence method to determine the (i). Morphological changes in nuclei structures, (ii). Assessing the apoptotic insults, (iii). Easy method to determine the cell viability and (iv). To distinguish the viable apoptotic and necrotic cells from early and late stage, usually applied to measure the apoptosis in cancer cells (4). In this paper we have proposed the AO/EB double staining test as a standard assay to assess the apoptotic potential of phenols. R. sativus L. plantlets were chosen as a model, because of their known sensitivity to several phytotoxic compounds and radish is a target specie in allelopathy studies (2). As a source of allelochemicals, we have tested the Olive Mill Wastewaters (OMWW) for their high content of polyphenols, the most common allelopathic agents (1). Besides, the apoptotic potential of catechol [the major component of OMWW phenolics (6)], has been also assessed, as it is considered responsible for the phytotoxicity of OMWW, reducing germination rates and seedlings development of cultivated plants and inhibiting the growth of soil and water microorganisms (3).

MATERIALS AND METHODS I. Origin of OMWW OMWW were collected from a Mill located in Puglia, Italy and stored in dark at 20°C. Biochemical composition has been previously reported (6). Based on the preliminary results, 4-concentrations (12.5, 25, 50, 100 % v/v) were prepared. Catechol was purchased from SIGMA (CAS# 120-80-9) and tested at concentrations of 10-4 to 10-3 M. All the test solutions were stirred in dark at room temperature for 48h and then stored at 4°C in dark for 2 weeks. Radish seeds were germinated under sterile conditions on filter paper disks embedded with distilled water. At 2 true leaves stage, radish seedlings were placed in plastic tubes, so that their root tips were immersed in test solution concentration, for 2 and 24 h (at 25°C with 16h-8h photoperiod). For each dose, 10-plantlets were processed. In control tests seedlings root tips were immersed in buffered distilled water. II. Ethidium bromide/acridine orange double staining assay The effects of OMWW on radish cells apoptosis were determined morphologically by fluorescent microscopy, after labelling with acridine orange and ethidium bromide. Acridine orange penetrates into the living and dead cells emitting green fluorescence [when intercalated into double strand nucleic acid (DNA)] and red fluorescence [when bound with single strand nucleic acid, such as RNA]. Ethidium bromide emits red fluorescence by intercalation into DNA, when cells have altered the cell membranes (4). Four kind of cells can be detected as per the fluorescence emission and the morphological aspect of chromatin condensation in the stained nuclei: (i). Uniform

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bright green nuclei, with organized structure, (ii). Early apoptotic cells with irregularly structured green nuclei, but chromatin condensation is visible as bright green patches or fragments or apoptotic bodies, (iii). Late apoptotic cells with orange to red nuclei with highly fragmented chromatin and (iv). Uniformly orange to red nuclei with organized structure, ascribable to necrotic cells. Root tips were cut from the radish seedlings and the plant tissues were digested by Polytron homogenizer placing the root tips in a lysis buffer (2.5M NaCl, 100mM Na2EDTA, 10mM Tris base, 1% TritonX, 1% DMSO). Cell suspension were collected by centrifugation, washed thrice with PBS and then resuspended in 25 µl of dye mixture [100 µg/ml of acridine orange and 100 µg /ml of ethidium bromide prepared in PBS and mixed gently]. Ten µl were placed on a microscope slide, covered with a coverslip and examined under 400 X magnification using the epifluorescence and filter combination (Nikon Eclipse E 800). Viable cells were identified by bright green nuclei with intact cells; early apoptotic cells were identified by nuclear condensation of chromatin as dense green patches or fragments. Late apoptotic and necrotic cells were identified by uniform labelling of cell nuclei, but apoptotic cells contained condensed chromatin or fragmented chromatin and were orange coloured and the necrotic cells showed orange nuclei with intact structures. III. Statistical analysis Statistical analyses were performed on all collected data. Data from EB/AO test were represented as percentage of live, apoptotic and necrotic cells. A minimum of 100 cells were counted for each treatment and control. To determine significant differences among each treatment group and the control group, data were assessed by one-way ANOVA when groups were homogeneous, and by ANOVA Kruskall-Wallis when groups were not homogeneous (SPSS Software Inc., Chicago). When significant differences (P < 0.05) appeared, Dunnet’s multiple comparison test was used to isolate the group(s) apart from a control group.

RESULTS AND DISCUSSION In this study we used the AO/EB double staining test, to evaluate the apoptotic potential of polyphenols on root cells of R. sativus. OMWW concentrations > 25% caused blackening of the radish root tips, thus preventing the evaluation of apoptosis, while at concentration < 12.5% the effects on radish radicle cells were negligible. The results obtained with AO/EB are shown in Figs 1, 2 and 3. All nuclei in control test (appearing as green in epifluorescence) showed a regular spherical structure and chromatin organization. The nuclei of root cells treated with OMWW were characterized by typical apoptotic hallmarks, clearly visible also in black and white pictures; the ongoing apoptotic process started with the loss of nuclear shape and the expansion of nuclear material (Fig. 1b). Progressively, several nuclear apoptotic bodies containing the fragmented DNA were visible, appearing as green fluorescent patches (early apoptosis, Fig. 1 c-d, arrow); in an advanced apoptotic stage the disintegration of apoptotic bodies lead to total nuclear DNA fragmentations typical of late apoptosis (orange to red stained nuclei, Fig. 1e, arrows). These data confirm that apoptotic-like bodies are fundamental marks of apoptosis in plants and in animals (5).

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Figure 1. The type of cell damage in radish roots revealed by AO/EB double staining test (Acridine orange/ethidium bromide). (a) Control nucleus; b-e) early apoptotic nuclei, with different levels of damage; (b) Condensed chromatin as dense patches (arrow); first stages of DNA fragmentations (c, d); e) Late apoptosis, with strongly fragmented chromatin.

In radicle cells treated with 25% OMWW dilutions, major DNA fragmentations and hypersegmented nuclei were observed (Fig. 1e) and a growing percentage of total apoptotic cells were registered during 24h (Fig. 2a, 85 to 98%). On the contrary, radish


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radicle cells treated with lower dose of OMWW (12.5%) showed a decrease in percentage of total apoptotic cells during 24h (Fig. 2b, 92 to 60%), due to significant decrease in early apoptotic cells (from 42 to 31%, Fig. 2b), while late apoptotic cells remained stable (around 50%, Fig. 2b) and a concomitant and relevant increase in cell viability (3.4 to 32%, data not shown). These findings confirmed that the apoptotic pathway in plant cells could be reversible at specific stages (2); in particular, early repairing mechanisms prevents the cell death, when the genetic damage produce only chromatin condensation and cells recover to divide. To confirm that the increase in cell viability observed in early stages of radish OMWW treatment is due to reversibility of chromatin condensation or DNA breaks, cytofluorometric investigations are required. OMWW 12.5%

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Figure 2. Different subsets of apoptotic cells as percentages of all cells measured by AO/EB fluorescence staining. Radish root cell treated for 2 h and for 24 h with 25% and 12.5% OMWW concentrations showed a time and dose-dependent effect in percentage of early (EA), late (LA), total (TA) apoptosis. (P< 0.001)

Previous works have demonstrated that catechol is one of the most active compounds present in olive mill wastewaters (2) and its concentration is 4x10-3 M (1), but all other polyphenols have lower concentration. According to these data, catechol concentrations in 25% and 12.5% OMWW correspond to 10-3 M and 5x10-4 M,

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respectively. The experiments with catechol were done at concentrations ranging from 10-3 M and 10-4 M. Catechol at concentration 10-3 M increased the nuclear damage during 24h, compared with control (62 to 90% total apoptotic cells, Fig. 3), thus suggesting that it could be mainly responsible for genotoxicity of OMWW. 100 90 80 70 60 50 40 30 20 10 0

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Figure 3. Different subsets of apoptotic cells as percentages of all cells measured by AO/EB fluorescence staining. Radish root cell treated for 2 h and for 24 h with three different concentration of catechol showed a time and dose-dependent effect in percentage of early (EA), late (LA), total (TA) apoptosis. (P< 0.001)

Catechol concentration of 5x10-4 M (corresponding to 12.5% OMWW) also have strong apoptotic potential during 24 h treatment, cells with typical apoptotic hallmarks strongly increased from 38% to 75% (Fig.1 c,d; Fig. 2 a, b). The lowest catechol test dose (10-4 M) also induced the alteration in nuclear morphology; fluoresced green and chromatin fragments were also visible, as in early apoptosis signals (Fig. 1 c). Catechol, therefore, was one of the OMWW phenolic compounds mainly involved in causing apoptosis in radish root cells. However, the discrepancy in results among 12.5% OMWW


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and 5x10-4 M catechol (ie decreasing vs increasing of apoptotic cells during 24 h treatment) could be explained by supposing that the simultaneous presence of different phenols in OMWW could determine an opponent or antagonist effect, thus reducing or, in some case, amplifying total toxicity (7, 8). Add Fig. 3 Morphologically programmed cells death, or apoptosis, is generally characterized by a series of modifications such as chromatin and cytoplasm condensation. Some reports suggest that features of plant apoptosis (such as nuclear disintegration and chromatin condensation triggered endogenously or environmentally) are similar to those in animals (15), while other features such as nuclear periphery and the formation of apoptotic bodies have not been universally identified. There is much evidence that polyphenols are phytotoxic and are involved in allelopathy; their role as preapoptotic compounds, already reported for cancer cells (5), has been also seen in plant cells. Our results indicate that AO/EB double staining assay can be recommended as a reliable and rapid assay to detect the apoptotic effect of allelochemicals in plant cells, since it allows distinction among the viable, early or late apoptotic cells, based on nuclear morphology variations and chromatin disintegrations. This method can be easily used in different plant species as well in any kind of plant tissue. To confirm these findings, further investigations using other plant model are in progress.

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