Cu(Ⅱ), Fe(Ⅲ) and Mn(Ⅱ) combinations as environmental stress factors have distinguishing effects on Enterococcus hirae

Pollution by various heavy metals as environmental stress factors might affect bacteria. It was established that iron(Fe(Ⅲ)), manganese(Mn(Ⅱ)) and copper(Cu(Ⅱ)) ion combinations caused effects on Enterococcus hirae that differed from the sum of the effects when the metals were added separately. It was shown that the Cu2+–Fe3+combination decreased the growth and ATPase activity of membrane vesicles of wild-type E. hirae ATCC9790 and atp D mutant(with defective FoF1-ATPase) MS116. Addition of Mn2+–Fe3+combinations within the same concentration range had no effects on growth compared to control(without heavy metals). ATPase activity was increased in the presence of Mn2+–Fe3+, while together with0.2 mmol/L N,N′-dicyclohexylcarbodiimide(DCCD), ATPase activity was decreased compared to control(when only 0.2 mmol/L DCCD was present). These results indicate that heavy metals ion combinations probably affect the FOF1-ATPase, leading to conformational changes. Moreover the action may be direct or be mediated by environment redox potential.The effects observed when Fe3+was added separately disappeared in both cases, which might be a result of competing processes between Fe3+and other heavy metals. These findings are novel and improve the understanding of heavy metals ions effects on bacteria,and could be applied for regulation of stress response patterns in the environment.     

Oxidative properties of ambient PM2.5 and elemental composition: Heterogeneous associations in 19 European cities

We assessed the extent to which constituents of PM_2.5 (transition metals, sodium, chloride) contribute to the ability to generate hydroxyl radicals (OH) in vitro in PM_2.5 sampled at 20 locations in 19 European centres participating in the European Community Respiratory Health Survey. PM_2.5 samples (n = 716) were collected on filters over one year and the oxidative activity of particle suspensions obtained from these filters was then assessed by measuring their ability to generate OH in the presence of hydrogen peroxide. Associations between OH formation and the studied PM constituents were heterogeneous. The total explained variance ranged from 85% in Norwich to only 6% in Albacete. Among the 20 centres, 15 showed positive correlations between one or more of the measured transition metals (copper, iron, manganese, lead, vanadium and titanium) and OH formation. In 9 of 20 centres OH formation was negatively associated with chloride, and in 3 centres with sodium. Across 19 European cities, elements which explained the largest variations in OH formation were chloride, iron and sodium.     

Effect of humic acids on physicochemical property and Cd(II) sorption of multiwalled carbon nanotubes

Zinc pyrithione is used as an antifouling agent. However, the environmental impacts of zinc pyrithione have recently been of concern. Zinc induces diverse actions during oxidative stress; therefore, we examined the effect of zinc pyrithione on rat thymocytes suffering from oxidative stress using appropriate fluorescent probes. The cytotoxicity of zinc pyrithione was not observed when the cells were incubated with 3 μM zinc pyrithione for 3 h. However, zinc pyrithione at nanomolar concentrations (10 nM or more) significantly increased the lethality of cells suffering from oxidative stress induced by 3 mM H₂O₂. The application of zinc pyrithione alone at nanomolar concentrations increased intracellular Zn²⁺ level and the cellular content of superoxide anions, and decreased the cellular content of nonprotein thiols. The simultaneous application of nanomolar zinc pyrithione and micromolar H₂O₂ synergistically increased the intracellular Zn²⁺ level. Therefore, zinc pyrithione at nanomolar concentrations may exert severe cytotoxic action on cells simultaneously exposed to chemicals that induce oxidative stress. If so, zinc pyrithione leaked from antifouling materials into surrounding environments would be a risk factor for aquatic ecosystems. Alternatively, zinc pyrithione under conditions of oxidative stress may become more potent antifouling ingredient.     

Impact of genetic diversity and inbreeding on the life-history of Chironomus midges over consecutive generations

We report results of a multigenerational experiment with Chironomus riparius. Two strains with a high and a low level of genetic variability were exposed to a low, environmentally relevant TBT concentration of 80 μg Sn kg⁻¹ sediment dw nominally (time weighted mean, based on measured concentrations: 4.5 μg Sn kg⁻¹ sediment dw), and various life history traits as well as genetic diversity were monitored for eleven consecutive generations. While TBT effects are hardly visible in the outbred and genetically diverse strain, the inbred and genetically impoverished strain shows a clearly reduced population growth rate compared to the control. Moreover, the impoverished strain shows an increase in fitness over time. Analyses of variation at five microsatellite loci revealed that the level of genetic variation is strongly reduced in the inbred compared to the outbred strain. Moreover, genetic diversity increases over time in the inbred strain. This finding explains the observed increase in fitness in both inbred lineages (control and TBT exposed). The results document that inbreeding and the level of genetic diversity might be of crucial importance in populations under pollution stress. Furthermore, ecotoxicological bioassays have to consider genetic diversity if results between laboratories should be comparable. Our data provides evidence that genetic diversity strongly contributes to the survival of a population exposed to chemical pollution.     

Short-term exposure of the European sea bass Dicentrarchus labrax to copper-based antifouling treated nets: Copper bioavailability and biomarkers responses

We studied if the levels of copper released from antifouling treated nets used in finfish mariculture could affect the immune defense mechanism and/or induce oxidative stress in Dicentrarchus labrax, after short term exposure in laboratory experiments. Dissolved copper concentration released from the treated nets, copper bioavailability and a set of biomarkers responses were measured. Biomarkers included hemoglobin concentration, activities of lysozyme, total complement, respiratory burst, glutathione S-transferase and acetycholinesterase and concentration of thiobarbituric acid reactive substances. Results indicated elevated copper concentration in seawater (184 μg L⁻¹) but low concentration in muscle (1.5 μg g⁻¹) and liver (117 μg g⁻¹). Copper bioavailability was independent of copper complexes with dissolved organic carbon. However, formation of copper complexes with other matrices could neither be excluded nor justified. The released copper from the treated nets did not induce oxidative stress but affected the immediate immune defense mechanism of the exposed fish making them more easily vulnerable to diseases. Consequently, copper-based antifouling treated nets could be a risk factor for D. labrax health.     

Atrazine promotes biochemical changes and DNA damage in a Neotropical fish species

The effects of Atrazine, an herbicide used worldwide and considered as a potential contaminant in aquatic environments, were assessed on the Neotropical fish Prochilodus lineatus acutely (24 and 48 h) exposed to 2 or 10 μg L⁻¹ of atrazine by using a set of biochemical and genetic biomarkers. The following parameters were measured in the liver: activity of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD) and glutathione S transferase (GST), antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), content of reduced glutathione (GSH), generation of reactive oxygen species (ROS) and occurrence of lipid peroxidation (LPO); in brain and muscle the activity of acetylcholinesterase (AChE) and DNA damage (comet assay) on erythrocytes, gills and liver cells. A general decreasing trend on the biotransformation and antioxidant enzymes was observed in the liver of P. lineatus exposed to atrazine; except for GR, all the other antioxidant enzymes (SOD, CAT and GPx) and biotransformation enzymes (EROD and GST) showed inhibited activity. Changes in muscle or brain AChE were not detected. DNA damage was observed in the different cell types of fish exposed to the herbicide, and it was probably not from oxidative origin, since no increase in ROS generation and LPO was detected in the liver. These results show that atrazine behaves as enzyme inhibitor, impairing hepatic metabolism, and produces genotoxic damage to different cell types of P. lineatus.     

Cornmeal-induced resistance to ciprofloxacin and erythromycin in enterococci

Triclosan is used as an antibacterial agent in household items and personal care products. Since this compound is found in maternal milk of humans and bodies of wild animals, there is growing concern among some consumer groups and scientific community that triclosan is adverse for humans and wild animals. In order to estimate adverse actions of triclosan, the effects of triclosan on intracellular Zn²⁺ concentration and cellular thiol content were studied in rat thymocytes by the use of flow cytometer with appropriate fluorescent probes. Triclosan at 1-3 μM (sublethal concentrations) increased the intensity of FluoZin-3 fluorescence (intracellular Zn²⁺ concentration) and decreased the intensity of 5-chloromethylfluorescein (5-CMF) fluorescence (cellular thiol content). Negative correlation (r = −0.985) between triclosan-induced changes in FluoZin-3 and 5-CMF fluorescences was found. Removal of external Zn²⁺ did not significantly affect the triclosan-induced augmentation of FluoZin-3 fluorescence, suggesting an intracellular Zn²⁺ release by triclosan. These actions of triclosan were similar to those of H₂O₂ and triclosan significantly potentiated the cytotoxicity of H₂O₂. Therefore, the results may suggest that triclosan at sublethal concentrations induces oxidative stress that decreases cellular thiol content, resulting in an increase in intracellular Zn²⁺ concentration by Zn²⁺ release from intracellular store(s). Since recent studies show many physiological roles of intracellular Zn²⁺ in cellular functions, the triclosan-induced disturbance of cellular Zn²⁺ homeostasis may induce adverse actions on the cells.     

Effect of ingested titanium dioxide nanoparticles on the digestive gland cell membrane of terrestrial isopods

The aim of this study was to find out whether ingested titanium dioxide nanoparticles (nano-TiO₂) cause cell membrane damage by direct contact or by lipid peroxidation. We assessed lipid peroxidation and digestive gland cell membrane stability of animals fed on food dosed with nano-TiO₂. Conventional toxicity measures were completed to determine if cellular effects are propagated to higher levels of biological complexity. An invertebrate model organism (Porcellio scaber, Isopoda, Crustacea) was fed with food containing nanosized TiO₂ and the result confirmed that at higher exposure concentrations after 3 d exposure, nano-TiO₂ destabilized cell membranes but lipid peroxidation was not detected. Oxidative stress as evidenced by lipid peroxidation was observed at longer exposure durations and high exposure doses. These data suggest that cell membranes are destabilized by direct interactions between nanoparticles and cell membrane, not solely via oxidative stress.     

How Plants Cope with Foreign Compounds. Translocation of xenobiotic glutathione conjugates in roots of barley (Hordeum vulgare) (9 pp)

Background, Aim and Scope Numerous herbicides and xenobiotic organic pollutants are detoxified in plants to glutathione conjugates. Following this enzyme catalyzed reaction, xenobiotic GS-conjugates are thought to be compartmentalized in the vacuole of plant cells. In the present study, evidence is presented for long range transport of these conjugates in plants, rather than storage in the vacuole. To our knowledge this is the first report about the unidirectional long range transport of xenobiotic conjugates in plants and the exudation of a glutathione conjugate from the root tips. This could mean that plants possess an excretion system for unwanted compounds which give them similar advantages as animals. Materials and Methods: Barley plants (Hordeum vulgare L. cv. Cherie) were grown in Petri dishes soaked with tap water in the greenhouse. - Fluorescence Microscopy. Monobromo- and Monochlorobimane, two model xenobiotics that are conjugated rapidly in plant cells with glutathione, hereby forming fluorescent metabolites, were used as markers for our experiments. Their transport in the root could be followed sensitively with very good temporal and spatial resolution. Roots of barley seedlings were cut under water and the end at which xenobiotics were applied was fixed in an aperture with a thin latex foil and transferred into a drop of water on a cover slide. The cover slide was fixed in a measuring chamber on the stage of an inverse fluorescence microscope (Zeiss Axiovert 100). - Spectrometric enzyme assay. Glutathione S-transferase (GST) activity was determined in the protein extracts following established methods. Aliquots of the enzyme extract were incubated with 1-chloro-2,4-dinitrobenzene (CDNB), or monochlorobimane. Controls lacking enzyme or GSH were measured. - Pitman chamber experiments. Ten days old barley plants or detached roots were inserted into special incubation chambers, either complete with tips or decapitated, as well as 10 days old barley plants without root tips. Compartment A was filled with a transport medium and GSH conjugate or L-cysteine conjugate. Compartments B and C contained sugar free media. Samples were taken from the root tip containing compartment C and the amount of conjugate transported was determined spectro-photometrically. Results: The transport in roots is unidirectional towards the root tips and leads to exsudation of the conjugates at rates between 20 and 200 nmol min-1. The microscopic studies have been complemented by transport studies in small root chambers and spectroscopic quantification of dinitrobenzene-conjugates. The latter experiments confirm the microscopic studies. Furthermore it was shown that glutathione conjugates are transported at higher rates than cysteine conjugates, despite of their higher molecular weights. This observation points to the existence of glutathione specific carriers and a specific role of glutathione in the root. Discussion: It can be assumed that long distance transport of glutathione conjugates within the plant proceeds like GSH or amino acid transport in both, phloem and xylem. The high velocity of this translocation of the GS-X is indicative of an active transport. For free glutathione, a rapid transport-system is essential because an accumulation of GSH in the root tip inhibits further uptake of sulfur. Taking into account that all described MRP transporters and also the GSH plasmalemma ATPases have side activities for glutathione derivatives and conjugates, co-transport of these xenobiotic metabolites seems credible. - On the other hand, when GS-B was applied to the root tips from the outside, no significant uptake was observed. Thus it can be concluded that only those conjugates can be transported in the xylem which are formed inside the root apex. Having left the root once, there seems to be no return into the root vessels, probably because of a lack of inward directed transporters. Conclusions: Plants seem to possess the capability to store glutathione conjugates in the vacuole, but under certain conditions, these metabolites might also undergo long range transport, predominantly into the plant root. The transport seems dependent on specific carriers and is unidirectional, this means that xenobiotic conjugates from the rhizosphere are not taken up again. The exudation of xenobiotic metabolites offers an opportunity to avoid the accumulation of such compounds in the plant. Recommendations and Perspectives: The role of glutathione and glutathione related metabolites in the rhizosphere has not been studied in any detail, and only scattered data are available on interactions between the plant root and rhizosphere bacteria that encounter such conjugates. The final fate of these compounds in the root zone has also not been addressed so far. It will be interesting to study effects of the exuded metabolites on the biology of rhizosphere bacteria and fungi.     

Involvement of mitochondrial-mediated caspase-3 activation and lysosomal labilization in acrylamide-induced liver toxicity

Acrylamide (ACR) is a chemical frequently used in both industrial and synthetic processes and may be produced during food processing. ACR at very high concentrations is postulated to exert its toxicity through the stimulation of an oxidative stress. ACR in excessive doses induces the central nervous system, reproduction, and genetic toxicity. However, ACR effects on the liver, a major organ of drug metabolism, have not been adequately explored. In addition, the role of mitochondria in an ACR-mediated hepatotoxicity is still unclear. The aim of this study was to investigate the cytotoxic mechanisms attributed to ACR using isolated rat hepatocytes. Hepatocytes were isolated by the collagenase perfusion method and incubated with an EC50_2hr concentration of ACR for 3 hr. The EC50_{2 hr} of ACR on isolated rat hepatocytes was determined to be 1 mM. Based on our results, hepatocytes cytotoxicity of ACR (1 mM) was mediated by a reactive oxygen species formation and lipid peroxidation. Incubation of hepatocytes with ACR produced rapid hepatocyte glutathione depletion which is another marker of the cellular oxidative stress. ACR cytotoxicity was also associated with mitochondrial injury as evidenced by the decline of mitochondrial membrane potential and lysosomal membrane leakiness. Our results also showed that ACR induced caspase-3 activation, the final mediator of apoptosis signaling. These findings contribute to a better understanding underlying mechanisms involved in ACR hepatotoxicity originating from the oxidative stress and ending in mitochondrial/lysosomal damage and cell death signaling.