Genotoxic effects of Heracleum sosnowskyi in the Allium cepa test

Abstract

Heracleum sosnowskyi is one of the most aggressive alien species in Europe. However, no information is available on its genotoxic effects. The goal of this study was to investigate cytological aberrations and genotoxic effects of H. sosnowskyi juice using the well-established Allium test. Root tip cells were treated with varying concentrations (1, 5, 10, 50, 100 and 300 ml l^–1) of solution in water of H. sosnowskyi juice. The same slides with root tips were used for mitotic abnormalities, chromosome aberrations and micronuclei scoring. Mitotic and phase indexes were also analyzed. No genotoxic effects were found at a concentration of 1 ml l^–1. Mitotic abnormalities were detected at concentrations of 10, 50, 100, and 300 ml l^–1. Chromosomal abnormalities and micronuclei were detected at concentrations of 50, 100, and 300 ml l^–1. Significant decrease in mitotic index was found at 5, 10, 50, 100, and 300 ml l^–1 concentrations of H. sosnowskyi juice. Lagging chromosomes were the most common abnormalities. The data demonstrated that H. sosnowskyi juice exhibits both clastogenic and aneugenic activity. However, aneugenic activity is more pronounced.

Keywords:

• Heracleum sosnowskyi
• genotoxicity
• mitotic index
• nuclear budding
• chromosome aberrations
• micronuclei
• Allium test

1. Introduction

Currently Sosnowsky’s hogweed (Heracleum sosnowskyi Manden, family Apiaceae), together with giant hogweed, Heracleum mantegazzianum (H. mantegazzianum Manden), and Persian hogweed (H. persicum Manden), is an active invasive species in Europe. The alien range of H. sosnowskyi covers the Baltic States, European part of Russia, Belarus and Ukraine, sporadically occurring in Poland, Hungary, Germany and Denmark. However, detailed distribution data could not be found (Nielsen et al. 2005). H. sosnowskyi is included in the lists of the most aggressive alien species in all the Baltic States. It is also included in the European and Mediterranean Plant Protection Organization’s (EPPO) list of invasive alien plants. Giant hogweed has a serious negative impact on biodiversity, destroying natural ecosystems, causing significant economic damage and posing a danger to human health. It is important to know that H. sosnowskyi belongs to a group of toxic plants, dangerous both to humans and animals (Jakubowicz et al. 2012). However, only during the last few years has the community been informed about the negative impacts caused by H. sosnowskyi (Rzymski et al. 2015). Universal ways to eliminate them have not been developed, so these species require detailed study (Rzymski et al. 2015).

There is a well-known phototoxic effect of juice from H. sosnowskyi, H. mantegazzianum, and H. persicum. These plants produce a clear watery sap, which contains photosensitizing compounds furanocoumarins. When it is in contact with skin, and in the presence of ultraviolet radiation, these compounds are activated and cause burns. Furanocoumarins present in the sap of hogweeds can cause cancer (Nielsen et al. 2005). Sap extracts are also considered to have allelopathic activity (Jakubska-Busse et al. 2013).

The juice of different kinds of hogweed can have other negative properties. Experiments on brine shrimp (Artemia salina L.) revealed that the extracts of H. persicum and H. sphondylium have a strong toxic effect without photoactivation, and cause the death of aquatic organisms (Mosham et al. 2009). Another study found that the extract of H. sibiricum Manden. has a mutagenic effect in culture of mammalian lymphocytes (Bogucka-Kocka et al. 2008). Allelopathic activity for several Heracleum species (H. sosnowskyi, H. asperum, H. dissectum, H. lescovii, and H. hirtum) was reported (Mishyna et al. 2015). Despite the seriousness of the problem and the need for detailed toxicological and genotoxicological study of different plants species (especially invasive ones), these data remain lacking (Modallal et al. 2008; Abderrahman and Shbailat 2014; Karaismailoglu 2014; Falistocc 2016; Arshad et al. 2016). Therefore, extensive studies are required to understand the effect of H. sosnowskyi on human health and environment. There have been no genotoxicological studies regarding H. sosnowskyi.

Plant bioassays are important tests in the detection of genotoxic and toxic agents and contamination in the environment (Frescura et al. 2013; Dixit et al. 2013; Adamakis et al. 2013). Allium cepa L. has been used to evaluate DNA damage, such as chromosome aberrations, micronuclei and disturbances in the mitotic cycle (Bakare et al. 2012; Achary et al. 2013). Due to its sensitivity, the A. cepa test was the first of nine plant assay systems evaluated by the Gene-Tox Program of the US Environmental Protection Agency (Grant 1994). The Allium test is now frequently used for environmental monitoring (Leme and Marine-Morels 2009; Olorunfemi et al. 2012; Olorunfemi et al., 2015), laboratory studies (Pesnya and Romanovsky 2013; Pesnya 2013; Kibrik et al. 2013; Prokhorova et al. 2013; Ślusarczyk et al. 2014; Karaismailoglu 2016), nanomaterials (Kumari et al. 2009; Klancnik et al. 2011; Panda et al. 2011; Sobieh et al. 2016) and is also proposed for assessing plant extracts (Kuhn et al. 2015).

The aim of this study was to investigate the genotoxic effects of H. sosnowskyi juice.

2. Materials and methods

2.1. H. sosnowskyi juice preparation

Stems and leaves of H. sosnowskyi were used. Plants were collected from abandoned farms (Nekrasovskiy district of the Yaroslavl region, village Burmakino 57°26′00″ N, 40°18′00″ E). Fresh stems and leaves were homogenized and filtered to obtain juice. The juice was diluted with distilled water (pH = 6.7) for different concentrations. The following concentrations were used: 300, 100, 50, 10, 5, and 1 ml l^–1 (Celik and Aslanturk 2009). All preparations were immediately used in the Allium cepa test.

2.2. Allium test and treatment

The Allium test was used to analyze genotoxic activity (Constantin and Owens 1982; Fiskesjo 1985).

The onion bulbs (Allium cepa L., 2n = 16) were of the Stuttgarten-Risen variety, average weight 25 g. Bulbs of A. cepa were placed in small glass jars with their basal ends dipped in distilled water and germinated at room temperature (24 ± 3°C). When the newly emerged roots were 0.50 cm in length they were used in the test. Roots of A. cepa were treated with a series of concentrations of H. sosnowskyi juice, i.e. 300, 100, 50, 10, 5, and 1 ml l^–1 for 96 h in the absence of sunlight and direct light. Control groups were treated with deionized distilled water. After the treatment the root-tips were placed in a solution of ethanol (96%) and glacial acetic acid (3:1) for 48 h then washed with distilled water and dyed using aceto-orcein for 1 h. The squash technique was applied for the study of the mitotic index (MI) and phase indexes, mitotic and chromosomal aberrations and micronuclei. Five replicates (bulbs) were performed for each group and scoring was given from the three roots of each replicate (15 slides for each concentration) (Barbério et al. 2011). The MI was calculated for each treatment as a number of dividing cells per 700 cells and also scored the proportions of mitotic phases.

Chromosomal aberrations (chromatid (single) and chromosome (double) bridges and fragments) were scored in 100 ana-telophases per slide. Mitotic abnormalities (lagging chromosomes and polyploidy) were scored in 1000 mitotic cells per slide. Micronuclei frequency was expressed as the number of interphase cells with micronuclei per 3000 for every slide. All examinations were done under a light microscope at 400 magnifications. The most frequent abnormalities (Figures 1(B) and (C) and 2) and normal cells (Figure 1(A)) are shown in photomicrographs. The results regarding MI and phase indexes and the frequency of mitotic and chromosomal abnormalities in root-tip cells of A. cepa are summarized in Tables 1, 2 and 3.

Table 1. Data on mitotic and phase indexes (mean ± SD) in the meristematic cells of A. cepa roots treated with solutions of H. sosnowskyi juice.

Juice concentration (ml l^–1)	Mitotic index (%)	Phase index (%)
		Prophase	Metaphase	Anaphase	Telophase
Control (0)	10.7 ± 0.6	65.5 ± 1.8	15.0 ± 1.6	9.6 ± 1.8	9.9 ± 0.5
1	10.4 ± 0.8	65.6 ± 1.0	19.1 ± 1.5	7.0 ± 1.4	8.3 ± 0.9
5	8.1 ± 0.4^a	65.6 ± 3.2	18.8 ± 1.9	9.8 ± 2.3	15.6 ± 4.2
10	6.5 ± 0.1^a	50.9 ± 1.7^a	25.8 ± 2.9	11.0 ± 1.5	12.3 ± 2.0
50	5.1 ± 0.4^a	47.7 ± 1.7^a	27.9 ± 2.3^a	12.9 ± 2.4	11.5 ± 2.0
100	3.4 ± 0.8^a	56.3 ± 6.1^a	23.9 ± 6.2^a	8.2 ± 2.9	11.6 ± 4.6
300	1.8 ± 0.2^a	45.8 ± 0.6^a	28.0 ± 0.7^a	13.5 ± 1.0^a	12.7 ± 0.1^a

^a Statistically different when compared with untreated control.

Table 2. Data on the number of mitotic disturbances (mean ± SD) in the meristematic cells of A. cepa roots treated by H. sosnowskyi juice.

Juice concentration (ml l^–1)	Mitotic abnormalities (% per 1000 cells)
	Polyploidy	Lagging chromosomes	Total (%)
Control (0)	0.00	0.43 ± 0.17	0.43 ± 0.17
1	0.00	0.35 ± 0.12	0.35 ± 0.12
5	0.00	1.21 ± 0.64	1.21 ± 0.64
10	0.00	3.05 ± 1.22^a	3.05 ± 1.22^a
50	0.00	8.31 ± 0.59^a	8.31 ± 0.59^a
100	0.00	15.56 ± 1.70^a	15.56 ± 1.70^a
300	0.00	10.27 ± 1.35^a	10.27 ± 1.35^a

^a Statistically different when compared with untreated control.

Table 3. Data on the number of chromosomal aberrations and micronuclei (mean ± SD) in the meristematic cells of A. cepa roots treated with solutions of H. sosnowskyi juice.

Juice concentration (ml l^–1)	Chromosomal abnormalities (% per 100 ana-telophases)
	Bridges	Fragments	Total (%)	Micronuclei (% per 3000 cells)
Control (0)	0.60 ± 0.22	0.27 ± 0.10	0.87 ± 0.26	0.012 ± 0.006
1	0.52 ± 0.14	0.31 ± 0.17	0.83 ± 0.17	0.011 ± 0.002
5	1.02 ± 0.20	0.70 ± 0.41	1.72 ± 0.34	0.021 ± 0.010
10	1.50 ± 0.36	0.92 ± 0.14	2.42 ± 0.67	0.036 ± 0.019
50	2.77 ± 0.12^a	2.84 ± 0.71^a	5.61 ± 0.89^a	0.101 ± 0.031^a
100	5.64 ± 0.72^a	4.73 ± 0.91^a	10.37 ± 0.85^a	0.189 ± 0.024^a
300	3.22 ± 0.46^a	4.15 ± 0.23^a	7.37 ± 0.43^a	0.236 ± 0.051^a

^a Statistically different when compared with untreated control.

Figure 1. (A) Control meristematic cells of A. cepa; (B) destruction of interphase chromatin (nuclear budding) and formation of micronuclei in root meristem of A. cepa after incubation in solution of 100 ml l^–1 H. sosnowskyi juice; (C) micronuclei at the center in root meristem of A. cepa after incubation in solution of 100 ml l^–1 H. sosnowskyi juice.

Figure 2. Mitotic and chromosome abnormalities and micronuclei in root meristematic cells of A. cepa: bridges (A, B), fragments (C, D), lagging chromosomes (E, F), micronuclei (G, H), achromatic spindle disturbances at metaphase (J), normal metaphase (K).

2.3. Statistical analysis

Statistical calculations were done using Statistica 8.0 (Dell Software, https://software.dell.com/products/statistica/). The differences in the mitotic index and phases indexes between treated and control groups were tested applying the non-parametric Mann–Whitney test. Frequencies of chromosome aberrations, micronuclei and mitotic abnormalities were statistically analyzed by Student’s t-test and ANOVA. The level of significance was accepted at p ≤ 0.05 (*).

3. Results and discussion

3.1. Mitotic and phase indexes

H. sosnowskyi juice at concentrations of 5–3000 ml l^–1 significantly decreased the number of dividing cells in A. cepa root meristems. The most pronounced effect was registered at 300 ml l^–1 concentration (Table 1). It should be noted that the antiproliferative activity of other medicinal plants using the A. cepa test has already been observed in many studies (Leme Marine-Morels 2009; Frescura et al. 2012; Frescura et al. 2013; Kuhn et al. 2015). Additionally, phase indexes were affected at 10–300 ml l^–1 concentrations of H. sosnowskyi juice. The prophase index is decreased, while metaphase index is increased. Anaphase and telophase index are increased at a 300 ml l^–1 concentration of H. sosnowskyi juice (Table 1). According to Prokhorova et al. (2008), a decreasing frequency of prophases indicates that the percentage of cells entering into the first phase of mitosis is decreasing. This confirms the strong antiproliferative effect of H. sosnowskyi juice. The situation, when the frequency of metaphases increased can be associated with action of H. sosnowskyi juice on the achromatic spindle (Figure 2(J)). In this case chromosomal segregation cannot occur, which may result in the appearance of lagging chromosomes and genomic mutations (polyploidy, aneuploidy, etc.) (Prokhorova et al. 2008). For polyploidy occurrence, several mechanisms were proposed (Zimmet and Ravid 2000; Beyaz et al. 2013).

H. sosnowskyi juice at 1 ml l^–1 concentration did not cause statistically significant changes in MI and phase indexes (Table 1).

Our results indicate that H. sosnowskyi juice disturbs the proportions of mitotic phases, which may result in mitotic abnormalities.

3.2. Mitotic abnormalities

In the control root tip cells were registered lagging chromosomes with a frequency of 0.43 ± 0.17% (spontaneous level). H. sosnowskyi juice caused a significant increase in the frequency of lagging chromosomes at concentrations of 10, 50, 100 and 300 ml l^–1 (Table 2, Figure 2(E) and (F)). The most pronounced aneugenic effect was registered at a 300 ml l^–1 concentration of H. sosnowskyi juice (Table 2).

3.3. Chromosomal abnormalities and micronuclei

Spontaneous frequencies of chromosomal aberrations (bridges and fragments) were 0.60 ± 0.16 and for micronuclei 0.012 ± 0.006 (Table 3).

Treatments with 1, 5 and 10 ml l^–1 concentrations of H. sosnowskyi juice did not increase the frequencies of chromosomal abnormalities or micronuclei over the control values.

Significant increase in the frequency of chromosomal aberrations was observed only after exposure to 50 ml l^–1 of H. sosnowskyi juice. Bridges (Figure 2(A) and (B)) and fragments (Figure 2(C) and (D)) were registered in root meristems of A. cepa at this concentration. Additionally, a significant increase in frequency of micronuclei (Figure 2(G) and (H)) was registered (Table 3). Micronuclei may arise mostly from acentric fragments or lagging chromosomes (Fenech 2000). Fragments (Figure 2(C) and (D)) can be derived from chromosomal breakages caused by clastogenic effect or they may alternatively derive from chromosome aberrations, such as chromosomal bridges, which break up and originate acentric fragments (Fiskesjo 1993; Leme and Marin-Morales, 2008). Micronuclei may additionally be generated through nuclear budding in interphase (Lindberg et al. 2007).

The vast majority of chromosomal abnormalities constitute lagging chromosomes (15.56 ± 1.70%), indicating that the achromatic spindle is damaged or centromeric regions of chromosomes are damaged.

This is the first genotoxicological study on H. sosnowskyi showing that H. sosnowskyi juice has strong genotoxic activity and can induce mitotic abnormalities, chromosomal aberrations and micronuclei in plant cells. Additionally, nuclear budding in interphase and formation of micronuclei was registered (Figure 1(B) and (C)). This study shows that H. sosnowskyi exhibits genotoxic effects in vivo even without photoactivation.

Limited information about the chemical components of Sosnowski’s hogweed is available. It was believed that the most biologically active as well as hazardous components of hogweeds from different locations are various coumarins (Jakubska-Busse et al. 2013). Coumarins, as mentioned in earlier research include isoimperatorin, bergapten, psoralen, pangelin, angelicin, umbelliferone, isobergapten, isopimpinellin, marmezin, osthole, (+)-oxypeucedanin, pimpinellin, sphondin, and methoxalen (Abyshev and Denisenko 1973; Malikov and Saidkhodzahaev 2004). Malikov and Saidkhodzahaev (2004) described coumarins common to both H. sosnowskyi and H. mantegazzianum species: bergapten, methoxalen, angelicin, isobergapten, isopimpinellin, osthole, pimpinellin, sphondin and umbelliferone. Other early investigation of chemical composition of H. sosnowskyi showed 15 coumarins (3 oxicoumarins and 12 furanocoumarins) including psoralen, xanthotoxin, bergapten, and angelicin (Satsyperova 1984). A recent study showed that the five main furanocoumarins of H. sosnowskyi are xanthotoxin (1.15%), bergapten (1.04%), umbelliferone (0.83%), angelicin (0.63%) and sphondin (0.35%) (Yourlova et al. 2013). Genotoxic activity of xanthotoxin, bergapten and angelicin have been shown in early studies (Venturini et al. 1980; Schimmer 1981). On the other hand, antigenotoxic activity of umbeliferone has been shown (Rezaee et al. 2014). It is possible that the cytological aberrations and genotoxic effects of H. sosnowskyi juice observed in the present study is synergetic, i.e. the result of the combined activity of the all chemical compounds.

Several Heracleum species were already evaluated for their cytotoxic and anticancer activities. The apoptotic activity of H. sibiricum against human acute myeloblastic leukemia, human acute T cell leukemia, human eosinophilic leukemia, human Caucasian promyelocytic leukemia, human T cell leukemia lymphoblast, human T cell leukemia, human myeloma, human Caucasian normal B cell, and human T cell was studied (Bogucka-Kocka et al. 2008). Cytotoxic activities of four Heracleum species on Hela, LS180, and Raji cell lines were investigated. H. pastinacifolium and H. transcaucasicum exhibited strong activities. H. rechingeri showed activity only on LS180, while H. persicum did not show cytotoxicity (Firuzi et al. 2010). It was reported that the leaves of H. sprengelianum have strong toxicity against liver cancer cells and less toxicity toward normal cells (Sathak et al. 2014).

However, there have not been any epidemiologic or intervention studies on the genus Heracleum, and information about safety is not sufficient. All of the cytotoxicity studies on Heracleum species are in vitro. There is a lack of in vivo investigations on their cytotoxic and anticancer effects. Advanced studies are necessary to explore the mechanism of their cytotoxic effects on different models and especially on cancer cell lines, because many Heracleum species exhibited considerable anticarcinogenic activity (Bahadori et al. 2016).

4. Conclusions

The results of this study show that H. sosnowskyi juice did not exhibit genotoxicity at concentration 1 ml l^–1. Considerable genotoxic effects of H. sosnowskyi juice at concentrations of 10–300 ml l^–1 were observed. The mitotic index was significantly decreased at 5–300 ml l^–1 concentrations of H. sosnowskyi juice. Phase indexes were modified at 10–300 ml l^–1 concentrations of H. sosnowskyi juice. Formation of micronuclei via nuclear budding were detected at 100 ml l^–1 and 300 ml l^–1 concentrations of H. sosnowskyi juice. The total frequency of mitotic abnormalities (chromosome lagging), chromosome aberrations (fragments, bridges) and micronuclei were significantly increased at 10–300 ml l^–1 concentrations of H. sosnowskyi juice.

Thus H. sosnowskyi juice exhibits both clastogenic and aneugenic activity in plant cells. However, aneugenic activity is more pronounced.

Acknowledgments

The author would like to express gratitude to Vladimir B. Verbitsky, Tatiana S. Pesnya, Alexander S. Pesnya, Sergey E. Bolotov and Inna M. Prokhorova for their helpful advice and notes.

Disclosure statement

No potential conflict of interest was reported by the authors.