STRUCTURAL MODIFACATIONS IN CELL MEMBRANES OF BRYOPHYTA AMBLYSTEGIUM SERPENS AND BRACHYTHECIUM MILDEANUM AS INDICATOR OF POLLUTION BY HEAVY METALS OF WELL WATER ON THE TERRITORY OF PRECARPATHIANS Текст научной статьи по специальности «Биологические науки»

BIOLOGIA | БИОЛОГИЧЕСКИЕ НАУКИ

STRUCTURAL MODIFACATIONS IN CELL MEMBRANES OF BRYOPHYTA AMBLYSTEGIUM SERPENS AND BRACHYTHECIUM MILDEANUM AS INDICATOR OF POLLUTION BY HEAVY METALS OF WELL WATER ON THE TERRITORY OF PRE-

CARPATHIANS

I.V. Bryndzya

Lecturer of the Chair of Ecology and Geography Ivan Franko Drohobych State Pedagogical University

V.V. Grubinko Doctor of Biological Sciences, Professor, Ternopil Volodymyr Hnatyuk National Pedagogical University

ABSTRACT

The data for the cell membranes structure of Amblystegium serpens and Brachythecium mildeanum bryophyes from wells in Pre-Carpathian region depending on the water pollution with heavy metals is presented in the article. The phenomenon of doubling (multiplication) for the cell membranes of the studied species was detected, and the relationship between the heavy metals content in water and the bryophyta cells radius is proposed as a bioindicative index.

Key words: cell membrane, heavy metals, Amblystegium serpens, Brachythecium mildeanum, wells, Pre-Carpathians.

Formulation of the problem. Aquatic plants growing the absorption of metals from the environment by their surface

in places, where the sources of drinking water supply are [11]. Changes in the structural components of cell membranes

contaminated, suffer from toxic effects reducing their cleaning and aquatic plants' membranes may cause the onset of signal

capacity. However, their response to the effect of pollutants can for functional cells reconstruction under the influence of stress

be used for the study of adaptation to various factors, as well as factors [16, 17]. These changes are considered are specific,

the indicators of toxic pollution, including the effect of heavy correlated with the accumulation level of metals in cells and

metals. [13] can therefore be used to establish the risk of water pollution

Analysis of recent researches and publications. Bryophyta with heavy metals [11, 12].

growing in water absorb heavy metals from contaminated Purpose of the study The purpose of our study was to detect

water in much larger quantities comparing Tracheophytes, as changes in the formation of bryophytes' cell membranes in

the pollutants are absorbed by all plant's surface [11]. It was terms of heavy metal contamination ofwater from wells located

shown for some Carpathian bryophytes that the adaptive in the Pre-Carpathians territory.

changes in their organisms under influence of heavy metals are Materials and methods

expressed in morphological and structural changes and growth The objects of study were the water samples from wells

processes [6], changes in the metabolism, as well as at the located on the territory of the Pre-Carpathian region (Figure

membrane level, due to the action of specific mechanisms for 1).

Fig. 1 Schematic map of the studied area and sample locations: 1 4 - rural.

urbanized; 2 - technogenic; 3 - recreational;

The territory of Pre-Carpathians was nominally divided into four parts by the nature of anthropogenic load - technologically transformed, recreational, rural and urban area. The outskirts of Truskavets are the main objects of Carpathians natural reserve fund, and the wells located within this territory were identified as the recreation area. The territory of Drohobych and Stryi districts was identified as rural. Mykolaiv district, where the large enterprise (OJSC "Mykolaivtsement") is located, and the city Drohobych (where JSC "SPC - Galicia" is located) were identified as the technogenically tranformed area. The cities Boryslav, Drohobych, and Stryi were identified as the urbanized area.

All the surveyed wells had the concrete lining rings, and their depth to the water table varied from 5 to 12 m. Samples of water were taken from wells at a depth of 1-2 m with plastic samplers of 1 L volume.

The content of heavy metals in water was determined by atomic absorption spectrophotometry on the spectrophotometer C-115 M1 at wavelengths matching the absorption maximum for each of the analyzed metals. Before the analysis, the water samples were 5-10 times concentrated by vaporization.

The bryophytes were collected in July 2013. Cell membranes were extracted by Findley and Evans method [10]. The two-

phase system required for the extraction was prepared by mixing a solution of 0.25 M sucrose and 30% polyethylene glycol (PEG) solution with 0.2 M sodium phosphate solution and distilled water. The mixture was left to 24 hours at 4 °C, then the sediment was re-suspended in the upper phase prepared by previously described procedure. The resulting suspension is divided equally into three 50 mL polycarbonate tubes. 10 mL of lower phase was added into each of them, mixed and centrifuged at 2000 rpm. for 15 minutes in a horizontal rotor. The membrane material was taken with a syringe from the phase boundary. Painting of cell membranes and their microscopic examination was carried out by methods [15], staining with chlorine-zinc-iodine agent. A solution of the pigment was placed on slide to the drops of a solution containing isolated membranes, covered with cover glass and examined with 9000 magnification. Cells' dimensions were determined by linear measurement.

Statistical analysis for the data was performed using Statistica 5.5 software and Microsoft Office Excel 2007.

Results

Heavy metals (HM) are known to be among the most dangerous surface water and groundwater pollutants, as they are not exposed to destruction like organic substances and are constantly present in aquatic ecosystems. Their compounds

significantly affect the water quality and the functioning aquatic ecosystems [4]. Reaction activity of metal forms in the biological objects, metal concentration and forms of their presence in the aquatic environment affect the bioaccumulation and the degree of influence on the biota.

The total content of heavy metals in the water from wells was estimated in the studiy. It was compared with the standard parameters for the heavy metals content in the natural environment (Table 1).

Table 1

Heavy metals content in the water from wells on the Pre-Carpathian territory, ^g/L (M±m, n=5)

Wells Metals

Co Cu Fe Mn Ni Zn Cd Pb

Technogenically transformed 12.5±2.0 6.7±1.2 49.0±3.0 Trace 38.0±10.0 2.3±0.4 Trace Trace

Recreational 11.0±2.0 2.6±1.1 30.0±5.0 2.2±0.8 25.0±6.0 3.6±1.0 Trace Trace

Urban 16.0±4.0 5.5±1.4 38.0±10.0 1.4±0.4 5.0±1.00 9.3±2.0 Trace Trace

Rural 19.0±4.0 4.5±0.9 27.0±8.0 Trace 12.0±3.0 1.4±4.0 Trace Trace

In particular, it was established that the metal content in the water from wells in the studied areas was the highest for Fe, Ni and Co, the Cu and Zn content was almost one order of magnitude lower. Iron and copper content in the water from wells was the highest in the technologically transformed and urban areas, probably due to the contamination with surface runoff, and is smaller in the wells located in the agricultural and recreational areas. Despite the high iron content, its concentration in water does not exceed the background concentration and MPC, possibly because iron being extremely important for the aquatic organisms, is absorbed by them to a large extent [2].

As for the Cu content in water from wells in all areas, the concentration higher than background value was established, despite its concentration does not exceed the MPC value.

The zinc content was also within the normative values, because zinc as biogenic metal is actively absorbed by aquatic plants, in which zinc ions are involved in key photosynthesis reactions [8].

Mn is found in significant quantities only in water from wells in the urban and recreational areas. Manganese does not have a high complexing index [5] and its binding depends on the pH of water, the presence of organic substances and other complexing compounds, suspended components concentration and redox capacity of water [8].

Pb and Cd are found in water from wells only in trace amounts for all studied areas.

The overall metal content, particularly iron and nickel, as it

was established previously, is high in the water from wells in the urban and technologically transformed areas only in autumn and spring, due to the active decomposition of vegetative mass of plants, which can be a source of metals in the water [1]. Although the metal content is close to the MPC value only in the studied wells in the urban ecosystems, the content of metals in the water from wells in the rural area is the same as in the wells of technological and urban areas, and varies seasonally with the highest content of metals in the autumn-winter period and with a slight peak in July. It can cause changes in cell morphology of the bryophytes due to its chronic exposure.

The study of the species composition for the plants in the studied wells showed that the representatives of bryophytes are found there, namely: Creeping feathermoss (Amblystegium serpens) is found in the wells located in the rural area, Sand Feather-moss (Brachythecium mildeanum) is found in the wells located in technogenically transformed, recreational and urban areas.

In accordance with prevailing adaptation to their existence in the certain hydrological regime, the bryophytes have different mechanisms of accumulation and retention of metals. It was established that of the dead part of the moss cover has relatively high hydrolytic acidity, due to which the cells are characterized by a large absorption capacity and can retain the large quantities of both hydrogen ions and other chemical elements [7].

It was found that the certain changes in their cell membranes are common for all bryophytes (figure 2).

Fig. 2. Photomicrographies of Amblystegium serpens (rural area), Brachythecium mildeanum (technologically transformed, recreational and urban areas) cells x 9000; 1 - outer shell; 2 - inner shell.

It was found that the thickening of cell membranes depending on the concentration of heavy metals in water (Table 1) was observed in most cells. It is well known that the effect of different factors causing a cascade of compensatory-adaptive changes in the structure and composition of plant cell membranes resulting in a phenomenon of secondary membranes formation - structures found in many plant cells and located between the first membrane and plasma membrane [14].

The detected nature of thickening is correlated with the structural and functional alterations in the cell membrane under the influence of metal ions, which is showed by the authors [16, 17] in aquatic plants as a protective adaptation helping to normalize the cells function and metabolic activity and their survival under the influence of unfavorable factors. In addition, the significant morphological differences relating

to the changes in membrane thickness and cells size are detected in the cells grown in the medium containing toxic ions. Specifically, the cells acquire the second concentric circle of substances, cytoplasm graininess increases, the increase of vacuolization and condensation of white color compound is observed, the concentric shell thickens, and the area of nuclear-cytoplasmic space decreases. The authors [16, 17] state that the formation of "secondary concentric membranes" is the universal cells response to the toxic stress, and occurs during the first hours of stressor's action regardless its their nature.

Our studies have shown the formation of such a "double shell" system. The shell outer circle radius was the largest for the bryophytes from the urban area. It was 0.68 cm/9000 for the urban area; 0.39 cm/9000 for the technologically transformed area, 0.36 cm/9000 for the rural area, and 0.34 cm/9000 for the recreational area (Table 2).

Table 2

Morphometric parameters of Amblystegium serpens and Brachythecium mildeanum cells, цт, (M±m, n=3)

Cell parameters Area

transformed Technogenically Recreational Urban Rural

R 2.61±0.40 1.96±0.22 2.10±0.13 2.00±0.22

Ml 0.39±0.12 0.34±0.11 0.68±0.02 0.36±0.01

M2 1.66±0.16 0.89±0.32 1.39±0.05 1.57±0.04

Note: R - cell radius; Ml and M2 - thickness of the outer (primary) and internal (secondary) cell membranes circle respectively.

The of the cell membrane radius for Brachythecium mildeanum from wells located in the technologically transformed and urban areas is the highest, and it is correlated with the highest (apart from Co and Ni) concentration of heavy metals in the water in these areas. The increase of metal content over the period of study can be represented by the following series: Cd <Pb < Mn <Cu < Zn <Co< Ni <Fe.

Applying the Chaddock scale [3, 9] to present the correlation analysis results for the relationship between the cells radius and the metals concentration in bryophytes, the direct correlation for Cu (n = 3, r = 0.67), Ni (n = 3, r = 0.63), Fe (n = 3, r = 0.76) was established. The relationship for zinc and manganese content is absent. As for the relationship between the thickness of the outer circle or primary cell membranes and metals content, the correlation for Cu, Co and Zn (n = 3, r = 0.45, 0.5 and 0.81 respectively) was established. The direct correlation between the thickness of the inner or secondary circle of cell membranes and Cu, Fe and Co contents (n = 3, r = 0.8, 0.65 and 0.52 respectively) was also established. It must be noted that the average indexes including the effect of particular metal on bryophytes were found in the study, however, the possible consequences of their joint action were not considered.

The research results support the assumption that the effect of heavy metals results in the structural changes in the bryophytes cell membrane. The relationship between the metals concentrations and structural rearrangements of bryophytes cell membranes, without the joint action of metals, as well as the influence of other factors, is characterized by the uniformity of identified changes and indicates the cells sensitivity to metals and can serve as a risk parameter to biological objects.

Conclusions and proposals

The study supports the hypothesis that the metals cause adaptive changes in the cells morphology of the studied bryophytes. The extent of forming a double circle of substances in the Amblystegium serpens and Brachythecium mildeanum cell membranes is associated with biologically dangerous heavy metals content in the water from wells, most likely, by their joint action. The established relationship is explained by the active absorption of heavy metals by the bryophytes. The studied relationships can be used for qualitative assessment of the environmental hazard for the drinking water contaminated by metal compounds.

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ОСОБЛИВОСТ1 Д1АГНОСТИКИ МУТАЦ1ЙНОГО СТАТУСУ ГЕНА HER-2/NEU В КЛ1ТИНАХ РАКУ МОЛОЧНО1 ЗАЛОЗИ НА ПРИКЛАД1 УКРА1НСЬКО1 ПОПУЛЯЦП

Ж1НОК

KxiMyK Богдана Тараавна,

атрант кафедри промисловог бютехнологгг, НТУУ «Кигвський полтехшчний шститут» Дуган Олексш Мартем'янович, доктор бюлогЫних наук, професор, НТУУ «Кигвський полтехшчний шститут» Захарцева Любое Михайлiвна, доктор медичних наук, патологоанатомЫне вiддiлення, Кигвський онкологЫний диспансер Клименко Сергш Втторович, Нацюнальний науковий центр радiацiйног медицини НАМН Украгни, доктор медичних наук, професор, вiддiл медичног генетики 1ЕР FEATURES OF DIAGNOSTIC GENE MUTATION STATUS HER-2/NEU IN BREAST CANCER CELLS ON EXAMPLE OF UKRAINIAN WOMEN

Klimuk B.T., PhD student Department of Industrial biotechnology, National Technical University of Ukraine "Kyiv Polytechnic Institute

Dugan O.M., doctor of biological sciences, professor, National Technical University of Ukraine "Kyiv Polytechnic Institute Zahartseva L.M., doctor of medical, autopsy department, Kyiv Oncological Clinic

Klimenko S.V., doctor of medical, professor, State Institution National Research Center for Radiation Medicine of the National Academy of Medical Sciences of Ukraine

АНОТАЦ1Я

BHER-2/Neu онкоген, що кодуе трансмембранний рецептор тирозинкшази та схожий з рецептором етдермального фактора росту. Цей ген може амплiфiкуватися в клтинах раку молочног залози людини. У поточному до^дженш, були до^джет змши гена в 163 випадках раку молочног залози людини .

ABSTRACT

The HER-2/neu oncogene encodes a transmembrane tyrosine kinase receptor with extensive homology to the epidermal growth factor receptor. This gene has been shown to be amplified in human breast cancer cell lines. In the current study, alterations of the gene in 163 primary human breast cancers were investigated.

Ключовi слова: рак молочног залози, ген HER-2/neu, iмуногiстохiмiя, флуоресцентна гiбридизацiя in situ, Key words: Breast cancer, HER-2/neu gene, Immunohistochemistry, Fluorescence in situ hybridization.

Встановлено, що у жшок, як страждають на рак молоч-но1 залози (РМЗ), ген ИЕР-2/пеи може пвдвищувати свою актившсть, через що на поверхш пухлинних клггин збшь-шуеться кшьюсть рецепторiв ИЕЯ-2/пеи.

Ввжаеться необхвдним визначення експресп ИЕЯ-2/пеи в клйинах пухлини для ощнки прогнозу переб^у захво-рювання та ефективност хiмiотерапil у хворих на РМЗ [1]. Пперекспреая ИЕЯ-2/пеи виявляеться в 25-30% випадюв раку РМЗ [1], причому в 90-95% випадюв пперекспреая ИЕЯ-2/пеи е результатом амплiфiкацil гена ИЕЯ-2/пеи [2].

Бшок ИЕЯ-2/пеи е глжопротешом, за структурою подiбним до рецепторiв етдермального фактора росту. За внутршньопротокового пстотипу РМЗ частота його ви-

явлення становить 90%. Ген, що кодуе бшок, локалiзуeться в 17-й хромосомь його експресш виявляють у непух-линнш тканиш молочно1 залози. Ввдзначено, що за наяв-носта в клпинах рецепторiв стерощних гормошв та вщ-сутносл гшерекспресп HER-2/neu, незалежно вщ розмiру пухлини та стану лiмфатичних вузлiв, переб^ РМЗ бшьш сприятливий, а за вщсутносп рецепторiв стерощних гормошв i наявносп гшерекспресп HER-2/neu - бшьш агре-сивний.

В даний час видшеш два стандартизованих методи, що дозволяють звести до мшмуму помилки у визначенш експресп HER-2/neu: iмуногiстохiмiчний (1ГХ) та пбридиза-щя in situ (ISH), зокрема флуоресцентна (FISH).