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Krótkoczasowa ekspozycja glonów słodkowodnych na organiczne związki ołowiu – nieinwazyjne badania elektryczne i luminescencyjne

Borc R., Jaśkowska A., Dudziak A.

Inżynieria Ekologiczna

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2016

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Nr 50

26--35

PL

W pracy zastosowano dwie różne biofizyczne metody badawcze celem sprawdzenia czy są one dostatecznie czułe na wykrywanie absorpcji jonów metali ciężkich przez makrofity wodne (makroalgi Nitellopsis obtusa) w krótkim czasie po ich ekspozycji na działanie tych jonów. Obie techniki pomiarowe są całkowicie nieinwazyjne, dzięki czemu można je stosować in vivo. Pierwsza z tych technik to monitorowanie parametrów elektrycznych roślin za pomocą zmiennoprądowego mostka elektrycznego przy użyciu czterech elektrod zewnętrznych w roztworze (dwóch napięciowych i dwóch prądowych). Druga technika pomiarowa polega na rejestracji ultrasłabego (niewidzialnego gołym okiem) promieniowania elektromagnetycznego (UWL) z tych roślin w zakresie od bliskiego ultrafioletu poprzez przedział widzialny do bliskiej podczerwieni. Do badań testowych użyto dwóch organicznych związków ołowiu, tj. octanu ołowiu i chlorotrimetyloołowiu. Zmiany wartości parametrów elektrycznych jak i natężenia emisji fotonowej roślin świadczyły o poborze ołowiu przez rośliny z zanieczyszczonego środowiska. Możliwa była ocena czasu, po którym występowała absorpcja ołowiu powodująca zmiany w komórkach roślinnych na poziomie błon komórkowych, jak i oszacowanie tego poboru, który zależał od zastosowanego stężenia organicznych związków ołowiu. Wyniki badań pokazały interesującą zależność, a mianowicie, dla małych stężeń organicznych związków ołowiu w środowisku, tj. poniżej 1 mM, w czasie 5-cio godzinnej ekspozycji roślin na ich działanie, widoczne były wyraźne zmiany parametrów elektrycznych komórek roślinnych, podczas gdy ultrasłaba luminescencja tych roślin pozostawała na stałym poziomie. Przykładowo dla octanu ołowiu, potencjał spoczynkowy błony komórkowej w zakresie stężeń 0–100 mM zmieniał się od -140 mV do -175 mV, a rezystancja błony wzrastała 1,5 razy w stosunku do wartości wyjściowej dla stężeń od 12 do 50 mM. Dla stężeń związków ołowiu powyżej 1 mM, metoda elektryczna okazywała się mało przydatna, gdyż pojedyncze komórki ulegały szybkiemu załamywaniu się w trakcie pomiarów. Intensywność ultrasłabego „świecenia” roślin dla wyższych stężeń octanu ołowiu dochodzących do 10 mM wzrastała gwałtownie (nawet czterokrotnie) w okresie pierwszych 20 minut ekspozycji. Natomiast dla chlorotrimetyloołowiu efekt wzrostu UWL był bardzo łagodny i następował dopiero po około 40 minutach od momentu ekspozycji. Tak duże stężenia związków organicznych ołowiu w środowisku są czynnikiem wywołującym szok, stres organizmów roślinnych. Związany jest on najprawdopodobniej z gwałtowną erupcją wolnych rodników prowadzącą do peroksydacji lipidów i nieodwracalnymi zmianami letalnymi, jak sugeruje wielu autorów w literaturze naukowej. Szczegółowe badania biochemiczne w tym zakresie mogłyby pomóc w rozstrzygnięciu tego problemu.

EN

In this paper the non-invasive biophysical methods were applied to assess the organic lead compounds absorption by freshwater algae Nitellopsis obtusa. Two biophysical techniques were used: the electrical method AC bridge with four external electrodes and the luminescent with the registration of ultraweak photon radiation emitted by plants. They allow to study the electrical and luminescent cell membrane properties. Research was performed with the lead acetate and trimethyllead chloride to verify whether algae cells were able to absorb Pb ions from water medium contaminated by these compounds. When the concentration of lead acetate solution increased up to 0–100 mM the membrane resting potential changed from -140 mV to -175 mV. On the other hand, the electrical resistance of cell membrane (for 12–50 mM) increased with exposure time exceeding its starting value up to 1.5 times. In contrast to these electrical changes, the intensity of ultraweak luminescence was constant at concentrations below 1 mM lead acetate. These results with comparison of literature data can suggest that there was no lipid peroxidation in cell membranes for such organic lead compound concentrations. Chemiluminescent responses of algae were observed in the first 5 hours of experiments for lead concentration higher than 1mM. The luminescence intensity increased immediately for both reagents, but no more than 4 times for concentrations of 1–10 mM lead acetate. For tetramethyl-lead chloride, the luminescence intensity started to increase slowly about 40 minutes after injection. These results with comparison of literature data may suggest lipid peroxidation in cell membrane for higher, toxic lead concentrations. It means that higher concentrations of lead can trigger lethal processes in the living cells. We conclude that the return of the electrical and chemiluminescent plant parameters to the starting values (before the action of lead ions) can indicate whether living cells are able to cope with detoxifying from heavy metals and whether they can survive when exposed to certain concentration of lead compounds. Algae of Nitellopsis obtusa in the first stage of exposure (5 hours) are able to accumulate organic compounds of lead without essential perturbations only up to specified concentrations (for example 1 mM of lead acetate). For higher lead content, an increase of ultraweak luminescence occurred which was probably associated with free radical productions and lipid peroxidation, as many researchers suggested.

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Odbiór bodźców środowiskowych przez glony Characeae sygnalizowany za pomocą ultrasłabej luminescencji

Jaśkowska A., Dudziak A., Borc R

Rocznik Ochrona Środowiska

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2012

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Tom 14

380--392

EN

Protecting the environment requires ensuring such conditions that organisms living in it did not suffer permanent damage to their structures and life functions. This also applies to algae living in the aquatic environment. Some species of water flora especially quickly react to negative stimuli arriving from the environment. Their reactions to experienced stress may be a subtle criterion for assessing the condition of aquatic environment. To this kind of plants belong Chara, which are bioindicators of water quality. Their response to applied environmental stimuli is rapid changes of the electrical parameters of the cell membrane. Our research shows that there is also a change in the intensity of ultraweak luminescence (UWL) generated by these plants. UWL is a phenomenon that continuously accompanies processes occurring in living cells and its intensity is less than the threshold sensitivity of the human eye. The method of measurement of this little known way of Chara's response to environmental stimuli has the advantage that it is completely non-invasive for the test material. Non-invasiveness is due to the fact that the radiation emitted from the samples is measured outside the cell membrane separating the living matter from the surrounding environment. In our study, after exposure to a selected stimulus, three types of responses of plant cells were observed: 1) - after a temporary increase of UWL, there is a return to the level of light corresponding to undisturbed homeostasis (e.g. when ascorbic acid is added to the environment); 2) - after a temporary increase in the UWL, the emission of photons slightly falls and stabilizes at a new higher level of illumination (when the stress stimulus is e.g. lead acetate, the emission intensity is 2.2 times larger than in the beginning); 3) - after a sudden and significant increase of UWL, the high emission level is maintained for many hours (e.g. after adding sucrose follows a 20-fold increase of UWL). UWL curves obtained in our experiments correlate with the changes of entropy corresponding to the disturbance of homeostasis in individual cases, depending on the applied stimulus. Respectively: for 1) - an increase of entropy is temporary and is followed by a return to values before the application of the stimulus, 2) - a lasting change occurs, which consists in an increase of entropy to a new fixed level, 3) - change in entropy is so large that its value exceeds the so-called point of no return, which means that the disorder of homeostasis leads to cell death. The application of luminescence imaging technique Single Photon Counting Imaging (SPCI) to the test cells have revealed that the emission of photons from different parts of the plant has a different intensity. When the plant suffered mechanical damage by cutting pseudoleaves, the emission from the damaged sites increased dramatically. This observation allows the damage to be located. Thanks to SPCI technique, it is also possible to track over time any repair processes. The clear differentiation of UWL kinetics leads to the conclusion that monitoring its intensity can be a good research tool in environmental protection. It makes it possible to determine whether chemicals found in the aquatic environment are harmful to a given species of plants. It can be also assessed whether the degree of the harm threatens the entire population with extinction. Thus, the presented method can be used as an ecotoxicity test.

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Intermolecular interactions in plant cells reflected by ultraweak luminescence penomenon

Jaśkowska A., Dudziak A., Gospodarek M., Śpiewla E.

Materials Science Poland

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2006

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Vol. 24, No. 4

1115--1125

EN

It is commonly known that photons necessarily participate in atomic and molecular interactions. On the other hand, plant, animal and human cells spontaneously emit electromagnetic radiation. The universality of ultraweak cell luminescence is inherently associated with fundamental processes in living organisms. The majority of researchers think that this very weak radiation (10-105 photons/(cm2-s)) results from such radical reactions as, for instance, lipid peroxidation. Having at our disposal the spectra of ultraweak luminescence (UL) from intact Characeae cells and their particular cellular structures and fractions, we could confirm that the electron carriers of the mitochondrial respiration chain were active in cytoplasm and in the intact cells but not in the cell walls. We obtained a band of chlorophyll emission in UL spectra. The dark chlorophyll should not emit fluorescence, which we observed under illumination. Nevertheless in our experiments, in which plants were kept in darkness for ca. 12 hours prior to measurement, this emission was observed. The only way to account for this phenomenon is that the energy of excitation is transferred to chlorophyll by other molecules. In this ultraweak sensibilized luminescence, the excited carbonyl molecules 1,3(=C=O*) transfer their energy to chlorophyll molecules with a high quantum efficiency. As we found in the spectral analysis of UL, the intensity at the wavelength of 634 nm - corresponding to dimoles of singlet oxygen - markedly decreased when the cells were exposed to the action of ascorbic acid (AsA) and lignocaine, which are singlet oxygen quenchers.