ACCUMULATION OF HEAVY METALS AND ANTIOXIDANT DEFENSE SYSTEM IN THE GAMETOPHYTE OF DIDYMODON RIGIDULUS HEDW. WITH HIGH LOADS

control. The concentration of lipid hydroperoxides in the moss sampled at sites 2 and 3 was 4.26 and 3.75 times higher, respectively, compared to the control, and TBARS pro ­ duction was more intense in plant material from site 2 than from the control site. SOD and catalase activities were considerably increased in D. rigidulus moss from site 2 compared with those from the control area; however, the activity of both enzymes in the moss samples from site 3 did not significantly exceed the control levels. Conclusions. Both road and rail traffic loads contribute to the accumulation of heavy metals, especially Zn and Pb, in D. rigidulus moss growing in the surrounding areas. Under such conditions, LPO process is stimulated, which is more pronounced in moss growing in the area with heavy road traffic. The increased activity of antioxidant enzymes (SOD and catalase) in moss growing in this area can play an important role in protecting bryophyte cells against metal­induced oxidative stress under conditions of intense metal


INTRODUCTION
Various modes of transport are known to be among the main sources of environ mental pollution. In Ukraine, road transport accounts for an average of 34 % of the total releases of polluting substances from various sources, but in many regions emissions from motor vehicles prevail over emissions from stationary sources [3,19,30]. Of all the pollutants released into the atmosphere as a result of transport activities, heavy metals belong to the most hazardous ones [18,24]. Among the known mechanisms that medi ate the toxicity of heavy metals in biological systems is the formation of reactive oxygen species capable of interacting with cellular biomolecules. This is accompanied by stimu lation of the process of lipid peroxidation (LPO), inactivation of sulfhydryl groups of proteins, damage to cell membranes and nucleic acids [7]. Some groups of terrestrial vegetation, including bryophytes, are highly resistant to the toxicity of heavy metals and can accumulate these substances in large concentra tions [28]. Bryophytes are often used in the environmental assessment of the atmo sphere due to their ability to absorb pollutants, mainly from air and precipitation [17,21,28]. The resistance of mosses to metal toxicity is largely due to their physiological and metabolic characteristics [28]. However, the relationship between the intensity of metal uptake and the activity of defense systems in bryophyte cells has not been studied completely.
The aim of this study was to investigate the level of accumulation of heavy metals, intensity of LPO process and the state of the antioxidant system in the bryophyte Didymodon rigidulus Hedw. collected in areas of road and railway traffic loads within the territory of Lviv (Ukraine).

Study area.
In the course of the study, three separate sites were selected in areas with different levels of anthropogenic pressure within the city of Lviv. Site 1 was chosen in the central part of Sknylivskyi Park (49°48′55″N, 23°57′59″E) located in Zaliznychnyi District of the city. The park covers an area of almost 57.7 hectares; its central part is almost unaffected by technogenic factors. Therefore, this site was considered as a con trol plot. Sites 2 and 3 were selected in Frankivskyi District of Lviv, in areas subject to anthropogenic impact. Site 2 was chosen in the territory adjacent to Kulparkivska Street, near its junction with Okruzhna and Antonovycha streets (49°49′30.5″N, 23°59′23.4″E). Site 3 was selected at the end of Eugene Konovalets Street (49°49′23.5″N, 23°59′31.6″E) adjacent to the Lviv-Khodoriv railway line; this area is exposed to railway loads, but it is virtually unaffected by road traffic.
Analysis of moss material. The bryophyte Didymodon rigidulus is a member of the family Pottiaceae, class Bryopsida. It belongs to calciphilic species, which are dis tributed on substrates with a high content of calcium carbonate. The species is often found on partially shaded surfaces of rocks and stones, as well as on artificial sub strates containing calcite, such as concrete [9,27]. In natural ecosystems, D. rigidulus occurs on the surface of limestone and loess deposits, limestone blocks and soils. It has also been found as a component of the biological crust on the soil surface in arid and semiarid ecosystems [22]. In urban ecosystems, D. rigidulus can be found on old buil ding walls, limestone fences, lawns, sidewalks, and asphalt pavements. The species is common on the territory of Ukraine and other countries [4,9,10,27].
Gametophyte shoots were collected at sampling sites 1-3 and analyzed for metal content, levels of lipid peroxidation products and antioxidant system enzyme activities. Moss material was sampled in triplicate at each sampling site. Sampling and prepara tion of moss material for analysis were carried out using standard methods [29].
Concentrations of chromium (Cr), nickel (Ni), lead (Pb) and zinc (Zn) in moss game tophytes were determined by the method of atomic absorption spectrophotometry. The plant material was mineralized with HNO 3 and H 2 O 2 prior to analysis [17]. The measure ments were performed using a C-115PK Selmi atomic absorption spectrometer (Ukraine). Metal concentrations were expressed in milligrams per 1 kg of dry weight of the samples.
Analysis of the level of lipid hydroperoxides in moss tissues was carried out by a method based on spectrophotometric measurement (λ = 480 nm) of products formed in the tissue extracts in the presence of ammonium ferrous sulfate hexahydrate (Mohr's salt), hydrochloric acid, and ammonium thiocyanate [15]. Prior to reaction, proteins in homogenates were precipitated with trichloroacetic acid, and lipids were extracted with ethanol. To determine the concentration of thiobarbituric acid reactive substances (TBARS) in the moss material, the colorimetric method with thiobarbituric acid (TBA) was used [5].
The results were processed by methods of variation statistics [31]. Conducting a statistical analysis of the results, the arithmetic mean and its standard deviation (M ± S.D.) were calculated. When comparing data groups, the significance of differences was assessed using the Student's ttest. Differences between data groups obtained at each sampling site were considered significant at p <0.05.

RESULTS AND DISCUSSION
The results shown in Fig. 1, demonstrate the accumulative capacity of Didymodon rigidulus moss for four heavy metals, which are often found in high concentrations in the environmental components of industrial cities. It has been found that the content of Cr, Ni, Pb and Zn in the D. rigidulus gametophyte as a whole reaches values of the same order of magnitude as in other previously studied epilithic mosses, such as Schistidium apocarpum and Rhynchostegium murale [20]. Depending on the level of their accumu lation in D. rigidulus shoots, the analyzed metals can be arranged in the following order of decreasing concentration: Zn> Cr> Ni> Pb. However, the metal content in D. rigidulus varied according to the location of the sampling sites. The most significant differences were found in the concentrations of Pb and Zn in moss gametophytes collected at sites 2 and 3, compared with moss material from the park area (site 1). In particular, the con centration of Pb and Zn in D. rigidulus moss collected from Kulparkivska Street (site 2) was considerably higher (2.27 and 1.78 times, respectively, p<0.05) than in moss sam pled in Sknylivskyi Park (site 1). In the gametophyte of moss taken at site 3, the concentra tions of these metals were 1.8 and 1.67 times higher, respectively, compared to the levels recorded in moss collected at the control site. The concentration of Ni in D. rigidulus moss from sites 2 and 3 also showed an upward trend compared with plants collected from the park area, but the difference between the results was statistically significant (1.45 times, p<0.05) only for the moss sampled at site 2. The Cr concentration did not change in the moss collected from sites 2 and 3 as compared to the control. Elevated metal concentrations in the gametophyte of moss collected at sites 2 and 3 correspond to the intensity of traffic in these areas. In particular, Kulparkivska Street (site 2) belongs to the main thoroughfares of Lviv and is constantly loaded with passing cars, trucks and buses. According to our observations, the traffic intensity on this stretch of the street averages 1100 vehicles per hour in the daytime. Site 3 is located in close proximity to the railway line used for various types of rail transportation (passenger trains and freight trains). Taking into account the fact that both road and rail transport are potential sources of heavy metal pollution [6,12,34,36], the increased level of Zn, Pb and Ni accumulation in D. rigidulus moss can be associated with high concentrations of metals in atmosphere at sites 2 and 3. A number of other studies have shown ele vated levels of Zn, Pb, and other heavy metals in the soils of roadside and railway-side areas, as well as in tissues of vascular plants growing in the adjacent territories [6,12,33]. Studies conducted on terrestrial plants have shown that heavy metal pollution is often associated with an increased rate of formation of reactive oxygen species (ROS) and lipid peroxidation products in plant cells [2,23,25,26]. The results of this study indicate the activation of LPO processes also in D. rigidulus bryophyte collected in areas exposed to road and rail traffic loads (Fig. 2). Namely, gametophytes of D. rigidulus collected at site 2 were characterized by higher concentrations of lipid hydroperoxides and TBARS (4.25 and 1.48 times, respectively), and moss samples from site 3 had a higher content of lipid hydroperoxides (3.75 times) compared with those collected at the control site. However, no significant changes in the TBARS level in moss gametophytes collected at site 3 were observed as compared to the control. These data may indicate a lesser degree of meta bolic damage as a result of a lower level of metal accumulation in D. rigidulus shoots in the railway area compared to moss collected from area with heavy road traffic. Fig. 2. The level of lipid peroxidation products (lipid hydroperoxides and thiobarbituric acid reactive sub stances (TBARS)) accumulation in the gametophyte of Didymodon rigidulus collected in the studied areas in the territory of Lviv Comment: 1) in this and the following figures, data are expressed as a percentage of the control value (means ± S.D.); 2) *, ** statistically significant differences between samples taken at site 1 (control) and at sites 2 and 3 (* p <0.05; ** p <0.01)

Рис. 2.
Рівень накопичення продуктів пероксидного окиснення ліпідів (гідропероксиди ліпідів і речо вини, що реагують з тіобарбітуровою кислотою (ТБК-активні продукти)) в гаметофіті моху Didymodon rigidulus, зібраного на досліджуваних ділянках у м. Львові Примітки: 1) на цьому та наступному рисунках результати виражені у відсотках від контроль ного значення (M ± S.D.); 2) *, ** -статистично вірогідні відмінності між зразками, відібраними на ділянці 1 (контроль) і на ділянках 2 і 3 ( * -р <0,05; ** -р <0,01) It is known that under stressful conditions, including those associated with expo sure to heavy metals, the antioxidant defense system plays an important role in plant tissues [8,23,25,35]. Antioxidant enzymes and non-enzymatic components of the antioxidant system counteract the accumulation of LPO products in the cell compart ments and in the apoplast. The results of this study show that moss material taken at site 2 is characterized by an increased SOD and catalase activities (p <0.05) compared with moss gametophytes sampled in the park area (site 1) (Fig. 3). ) to hydrogen peroxide, which can subsequently decompose into water and oxygen mainly with the aid of catalase [14]. Consequently, these enzymes play an important role in protecting cells from the development of oxidative stress caused by the formation of ROS and LPO products. However, the activities of SOD and catalase in the moss material from sampling site 3 did not demonstrate any significant differences compared to the control. This indicates less pronounced metabolic changes in plants growing in the territory adjacent to the railway than in the area with heavy road traffic. At the same time, other components of the defense system may be involved in counteracting the development of oxidative stress in the gametophytes of moss growing at site 3. In addition to SOD and catalase, the detoxification of ROS and LPO products with the participation of nonenzymatic antioxidants and the activation of the glutathione system are also important links in the cellular defense mechanism [32]. All of these components of the antioxidant system together can participate in the protection of bryo phyte cells from metal toxicity and are important for ensuring their resistance to metal contamination in areas with high traffic loads.

CONCLUSIONS
Based on the results obtained, it can be concluded that both road and rail traffic loads contribute to the accumulation of heavy metals in the gametophytes of D. rigidulus growing in the adjacent territories. This is especially true for Zn and Pb, which inten sively accumulates in moss tissues in the conditions of environmental pollution by emis sions from both road and railway transport. The accumulation of metals in moss game tophytes is accompanied by a strong increase in the concentration of LPO products, which indicates the stimulation of ROS formation under the influence of heavy metals. Under these conditions, activation of antioxidant enzymes, such as SOD and catalase, can protect bryophyte cells against metalinduced oxidative stress. At the same time, the increased activity of antioxidant enzymes may play a role in the adaptation of D. rigidulus to heavy metal contamination in areas exposed to high traffic loads.

Conflict of interest:
The authors declare that the study was conducted in the absence of any commercial or financial relationship that could be construed as a poten tial conflict of interest.
Animal rights: This article does not include animal studies.

AUTHOR CONTRIBUTIONS
Сonceived