Petra Cvjetko

The effects of cadmium-zinc interactions on biochemical responses in tobacco seedlings and adult plants.

The objective of the present study was to investigate the effects of cadmium-zinc (Cd-Zn) interactions on their uptake, oxidative damage of cell macromolecules (lipids, proteins, DNA) and activities of antioxidative enzymes in tobacco seedlings as well as roots and leaves of adult plants. Seedlings and plants were exposed to Cd (10 µM and 15 µM) and Zn (25 µM and 50 µM) as well as their combinations (10 µM or 15 µM Cd with either 25 µM or 50 µM Zn). Measurement of metal accumulation exhibited that Zn had mostly positive effect on Cd uptake in roots and seedlings, while Cd had antagonistic effect on Zn uptake in leaves and roots. According to examined oxidative stress parameters, in seedlings and roots individual Cd treatments induced oxidative damage, which was less prominent in combined treatments, indicating that the presence of Zn alleviates oxidative stress. However, DNA damage found in seedlings, and lower glutathione reductase (GR) and superoxide dismutase (SOD) activity recorded in both seedlings and roots, after individual Zn treatments, indicate that Zn accumulation could impose toxic effects. In leaves, oxidative stress was found after exposure to Cd either alone or in combination with Zn, thus implying that in this tissue Zn did not have alleviating effects. In conclusion, results obtained in different tobacco tissues suggest tissue-dependent Cd-Zn interactions, which resulted in activation of different mechanisms involved in the protection against metal stress.

Thallium Toxicity in Humans

Thallium Toxicity in Humans Thallium is a naturally occurring trace element, widely distributed in the earths crust, but at very low concentrations. It does not have a known biological use and does not appear to be an essential element for life. It has been considered one of the most toxic heavy metals. Occasionally, there are reports on thallium poisoning as results of suicide or murder attempt or accident. The main threat to humans is through occupational exposure, environmental contamination, and accumulation in food, mainly in vegetables grown on contaminated soil. Increasing use in emerging new technologies and demanding high-tech industry constantly raise concern about exposure risk to all living organisms. Thallium is considered a cumulative poison that can cause adverse health effects and degenerative changes in many organs. The effects are the most severe in the nervous system. The exact mechanism of thallium toxicity still remains unknown, although impaired glutathione metabolism, oxidative stress, and disruption of potassium-regulated homeostasis may play a role. The lack of data about mutagenic, carcinogenic, or teratogenic effects of thallium compounds in humans calls for further research. Toksičnost Talija u Humanoj Populaciji Talij je u prirodi široko rasprostranjen teški metal, prisutan u vrlo niskim koncentracijama pa ga stoga ubrajamo u elemente u tragovima. Budući da organizmima nije potreban ni u jednoj razvojnoj fazi, ne ubrajamo ga u grupu esencijalnih elemenata. Talij zbog njegovih svojstava ubrajamo među najtoksičnije teške metale. Povremeno se još uvijek pojavljuju slučajevi u kojima je talij upotrijebljen kao sredstvo za pokušaj ubojstva, odnosno samoubojstva, ali i slučajevi nenamjernog, slučajnog trovanja talijem. U današnje vrijeme potencijalna opasnost od trovanja talijem postoji zbog profesionalne izloženosti, izbijanja ekološke katastrofe ili zbog akumulacije u hranidbenim lancima, uglavnom zbog uzgoja hrane na onečišćenom tlu. Sve češća uporaba talija u visokotehnološkoj industriji kao odgovor na zahtjeve moderne tehnologije neprestano povećava rizik od izloženosti svih živih organizama štetnim utjecajima talija u okolišu. Talij ima izuzetno negativan učinak na različite organske sustave, a osobito na živčani sustav. Mehanizmi toksičnosti talija još uvijek nisu u potpunosti razjašnjeni, premda važnu ulogu imaju poremećaji metabolizma glutationa, oksidativni stres i narušavanje homeostaze posredovane ionima kalija. Nedostatak podataka o mutagenim, kancerogenim ili teratogenim učincima talija i njegovih spojeva u ljudi opravdava buduća istraživanja ovog vrlo toksičnog metala.

Duckweed Lemna minor as a tool for testing toxicity and genotoxicity of surface waters

In this investigation growth parameters and certain endpoints (pigment content, peroxidase activity, lipid peroxidation and alkaline comet assay) were used to detect the toxic and genotoxic effects of surface water samples on duckweed plants. The surface waters of different origin and pollutant burdens were collected monthly over a 3-month monitoring period at three sampling sites along the river Sava and its confluents (Croatia). Physicochemical characterization of the water samples included measurements of conductivity, chemical and biological oxygen demand, levels of total suspended solids, nitrate, nitrite, ammonium, Kjeldahl nitrogen and orthophosphate. Surface water samples collected from three stations caused reduction of duckweed growth rates, chlorophylls and carotenoid contents and peroxidase activity. In contrast, damage to membrane lipids (estimated by malondialdehyde content) and especially to DNA (estimated by tail extent moment) markedly increased in duckweed exposed to industrial wastewater samples. The results from the study indicate the ability of selected biomarkers to predict the phyto- and genotoxic effects of complex water mixtures on living organisms as well as the relevance of duckweed as a sensitive indicator of water quality.

Ecotoxicological assessment of industrial effluent using duckweed (Lemna minor L.) as a test organism

This study aimed at assessing the toxic effects of industrial effluents using duckweed (Lemna minor L.) plants as a test system. Growth inhibition test according to standardized protocol (ISO 20079) was performed. The suitability of the Comet assay (indicates DNA damage) and certain parameters such as peroxidase activity and lipid peroxidation level, as biomarkers for environmental monitoring was evaluated. The water samples were collected monthly over a 3-month period from the stream near the industrial estate of Savski Marof, Croatia. All samples caused inhibition of growth rates based on frond number and biomass as well as decrease of chlorophylls content. In contrast, peroxidase activity, malondialdehyde content and tail extent moment (measure of DNA strand breaks) markedly increased. Obtained data demonstrate the relevance of duckweed as sensitive indicators of water quality as well as the significance of selected biological parameters in the reliable assessment of phyto- and genotoxic potential of complex wastewaters.

Toxicological characterization of the landfill leachate prior/after chemical and electrochemical treatment: A study on human and plant cells

In this research, toxicological safety of two newly developed methods for the treatment of landfill leachate from the Piškornica (Croatia) sanitary landfill was investigated. Chemical treatment procedure combined chemical precipitation with CaO followed by coagulation with ferric chloride and final adsorption by clinoptilolite. Electrochemical treatment approach included pretreatment with ozone followed by electrooxidation/electrocoagulation and final polishing by microwave irradiation. Cell viability of untreated/treated landfill leachate was examined using fluorescence microscopy. Cytotoxic effect of the original leachate was obtained for both exposure periods (4 and 24 h) while treated samples showed no cytotoxic effect even after prolonged exposure time. The potential DNA damage of the untreated/treated landfill leachate was evaluated by the comet assay and cytokinesis-block micronucleus (CBMN) assay using either human or plant cells. The original leachate exhibited significantly higher comet assay parameters compared to negative control after 24 h exposure. On the contrary, there was no significant difference between negative control and chemically/electrochemically treated leachate for any of the parameters tested. There was also no significant increase in either CBMN assay parameter compared to the negative control following the exposure of the lymphocytes to the chemically or electrochemically treated landfill leachate for both exposure periods while the original sample showed significantly higher number of micronuclei, nucleoplasmic bridges and nuclear buds for both exposure times. Results suggest that both methods are suitable for the treatment of such complex waste effluent due to high removal efficiency of all measured parameters and toxicological safety of the treated effluent.

OXIDATIVE STRESS AND DNA DAMAGE IN BROAD BEAN (VICIA FABA L.) SEEDLINGS INDUCED BY THALLIUM

Thallium (Tl) is a metal of great toxicological concern because it is highly toxic to all living organisms through mechanisms that are yet poorly understood. Since Tl is accumulated by important crops, the present study aimed to analyze the biological effects induced by bioaccumulation of Tl in broad bean (Vicia faba L.) as well as the plants antioxidative defense mechanisms usually activated by heavy metals. Thallium toxicity was related to production of reactive oxygen species in leaves and roots of broad bean seedlings following short-term (72 h) exposure to thallium (I) acetate (0, 0.5, 1, 5, and 10 mg/L) by evaluating DNA damage and oxidative stress parameters as well as antioxidative response. The possible antagonistic effect of potassium (K) was tested by combined treatment with 5 mg/L of Tl (Tl+) and 10 mg/L of potassium (K+) acetate. Accumulation of Tl+ in roots was 50 to 250 times higher than in broad bean shoots and was accompanied by increase in dry weight and proline. Despite responsive antioxidative defense (increased activities of superoxide dismutase, ascorbate peroxidase, and pyrogallol peroxidase), Tl+ caused oxidative damage to lipids and proteins as evaluated by malondialdehyde and carbonyl group levels, and induced DNA strand breaks. Combined treatment caused no oxidative alternations to lipids and proteins though it induced DNA damage. The difference in Tl-induced genotoxicity following both acellular and cellular exposure implies indirect DNA damage. Results obtained indicate that oxidative stress is involved in the mechanism of Tl toxicity and that the tolerance of broad bean to Tl is achieved, at least in part, through the increased activity of antioxidant enzymes.

Toxicity of silver ions and differently coated silver nanoparticles in Allium cepa roots

Silver nanoparticles (AgNPs) are the dominating nanomaterial in consumer products due to their well-known antibacterial and antifungal properties. To enhance their properties, different surface coatings may be used, which affect physico-chemical properties of AgNPs. Due to their wide application, there has been concern about possible environmental and health consequences. Since plants play a significant role in accumulation and biodistribution of many environmentally released substances, they are also very likely to be influenced by AgNPs. In this study we investigated the toxicity of AgNO3 and three types of laboratory-synthesized AgNPs with different surface coatings [citrate, polyvinylpyrrolidone (PVP) and cetyltrimethylammonium bromide (CTAB)] on Allium cepa roots. Ionic form of Ag was confirmed to be more toxic than any of the AgNPs applied. All tested AgNPs caused oxidative stress and exhibited toxicity only when applied in higher concentrations. The highest toxicity was recorded for AgNPs-CTAB, which resulted with increased Ag uptake in the roots, consequently leading to strong reduction of the root growth and oxidative damage. The weakest impact was found for AgNPs-citrate, much bigger, negatively charged NPs, which also aggregated to larger particles. Therefore, we can conclude that the toxicity of AgNPs is directly correlated with their size, overall surface charge and/or surface coating.

Effect of Copper on the Toxicity and Genotoxicity of Cadmium in Duckweed (Lemna minor L.)

Effect of Copper on the Toxicity and Genotoxicity of Cadmium in Duckweed (Lemna Minor L.) We investigated interactions between copper (in the concentrations of 2.5 μmol L-1 and 5 μmol L-1) and cadmium (5 μmol L-1) in common duckweed (Lemna minor L.) by exposing it to either metal or to their combinations for four or seven days. Their uptake increased with time, but it was lower in plants treated with combinations of metals than in plants treated with either metal given alone. In separate treatments, either metal increased malondialdehyde (MDA) level and catalase and peroxidase activity. Both induced DNA damage, but copper did it only after 7 days of treatment. On day 4, the combination of cadmium and 5 μmol L-1 copper additionally increased MDA as well as catalase and peroxidase activity. In contrast, on day 7, MDA dropped in plants treated with combinations of metals, and especially with 2.5 μmol L-1 copper plus cadmium. In these plants, catalase activity was higher than in copper treated plants. Peroxidase activity increased after treatment with cadmium and 2.5 μmol L-1 copper but decreased in plants treated with cadmium and 5 μmol L-1 copper. Compared to copper alone, combinations of metals enhanced DNA damage after 4 days of treatment but it dropped on day 7. In conclusion, either metal given alone was toxic/genotoxic and caused oxidative stress. On day 4 of combined treatment, the higher copper concentration was more toxic than either metal alone. In contrast, on day 7 of combined treatment, the lower copper concentration showed lower oxidative and DNA damage. These complex interactions can not be explained by simple antagonism and/or synergism. Further studies should go in that direction. Učinak bakra na toksičnost i genotoksičnost kadmija u vodenoj leći (Lemna minor L.) U svrhu istraživanja interakcija između bakra kao esencijalnog elementa te kadmija kao neesencijalnog i toksičnog metala, vodenu leću Lemna minor L. uzgajali smo na podlogama s kadmijem (5 μmol L-1) odnosno s bakrom (2,5 μmol L-1 i 5 μmol L-1) te s njihovim kombinacijama. Unos metala u biljke povećavao se s trajanjem pokusa, a kod kombinacije metala u biljkama je izmjerena niža količina kadmija nego u onima uzgajanima samo na kadmiju. U biljkama tretiranim pojedinačnim metalom došlo je do povećanja sadržaja malondialdehida (MDA) te aktivnosti katalaze i peroksidaze u odnosu na kontrolne biljke. Također, primijećeno je oštećenje DNA iako kod bakra tek sedmog dana tretmana. Količina MDA i aktivnost obaju enzima dodatno se povećala na tretmanu kombinacijom kadmija i bakra (5 μmol L-1) nakon četvrtog dana pokusa, dok se količina MDA smanjila nakon sedmog dana kod kombinacije kadmija i 2,5 μmol L-1 bakra. U tim biljkama primijećena je i veća aktivnost katalaze, dok je aktivnost peroksidaze porasla na tretmanu kadmijem i 2,5 μmol L-1 bakrom, ali se smanjila na tretmanu kadmijem i 5 μmol L-1 bakrom. Oštećenje DNA koje je bilo veće kod kombinacije metala nakon četvrtog dana, osobito u usporedbi sa samim bakrom, smanjilo se nakon sedmog dana pokusa. Iz ovih rezultata može se zaključiti da su oba metala u istraživanim koncentracijama toksična i genotoksična za vodenu leću i da uzrokuju oksidacijski stres. Kadmij u kombinaciji s bakrom više koncentracije bio je toksičniji od pojedinačnih metala nakon četvrtog dana pokusa, dok su u biljaka tretiranih kombinacijom kadmija i bakra niže koncentracije toksični učinci bili manji. Budući da su primijećene interakcije vrlo kompleksne i ne uključuju samo antagonizam odnosno sinergizam potrebna su daljnja istraživanja.

Proteomics of heavy metal toxicity in plants

Summary Plants endure a variety of abiotic and biotic stresses, all of which cause major limitations to production. Among abiotic stressors, heavy metal contamination represents a global environmental problem endangering humans, animals, and plants. Exposure to heavy metals has been documented to induce changes in the expression of plant proteins. Proteins are macromolecules directly responsible for most biological processes in a living cell, while protein function is directly influenced by posttranslational modifications, which cannot be identified through genome studies. Therefore, it is necessary to conduct proteomic studies, which enable the elucidation of the presence and role of proteins under specific environmental conditions. This review attempts to present current knowledge on proteomic techniques developed with an aim to detect the response of plant to heavy metal stress. Significant contributions to a better understanding of the complex mechanisms of plant acclimation to metal stress are also discussed. Sažetak Biljke su neprestano izložene različitim čimbenicima abiotičkog i biotičkog stresa koji nepovoljno utječu na njihovu produktivnost. Teški metali kao česti zagađivači okoliša vrlo nepovoljno djeluju na sva živa bića, uključujući biljke, životinje i ljude. Poznato je da teški metali mogu mijenjati ekspresiju biljnih proteina. Proteine ubrajamo u biološki vrlo važne makromolekule čija je aktivnost u stanici izravno ovisna o posttranslacijskim modifikacijama, koje nije moguće pratiti na razini genoma. Stoga je nužno provoditi proteomska istraživanja kako bi se razotkrila prisutnost i uloga proteina u različitim vrstama okolišnog stresa. U ovom radu sažete su različite tehnike i metode istraživanja učinaka teških metala na biljni proteom, uključujući i sažet osvrt na složene mehanizme odgovora biljke na stres izazvan teškim metalima.

Cadmium and Zinc Induced Similar Changes in Protein and Glycoprotein Patterns in Tobacco (Nicotiana tabacum L.) Seedlings and Plants

The effects of 10 μmol L-1 and 15 μmol L-1 cadmium (Cd), a nonessential toxic element and 25 μmol L-1 and 50 μmol L-1 zinc (Zn), an essential micronutrient, on proteins and glycoproteins of Nicotiana tabacum L. seedlings and plants were investigated after exposure to each metal alone or to their combinations. Changes in only few polypeptides related to heavy metal treatments were observed in tobacco seedlings and leaves of adult plants, while the greatest change in total soluble protein pattern was observed in plant roots. Differences between control and treated tobacco tissues were more pronounced in the glycoprotein pattern, which was analysed by application of different lectins. The majority of the detected glycoproteins in leaves and roots of adult plants can be considered as a result of enhanced glycosylation due to heavy metal stress. The difference in glycoproteins between Cd and Zn application on tobacco seedlings and adult plants could not be determined since enhanced glycosylation was noticed after treatment with either metal alone or in combination. Therefore, it can be concluded that both metals induced N- and Oglycosylation as a result of changed environmental conditions.