pH-dependent toxicity of copper and uranium to a tropical freshwater alga (Chlorella sp.)

Copper (Cu) and uranium (U) are of potential ecotoxicological concern to tropical freshwater organisms in northern Australia as a result of mining activity. No local data on the toxicity of these metals to tropical freshwater algae are currently available. The aim of this study was to investigate the effect of pH (5.7 and 6.5) on the toxicity of Cu and U to the green alga Chlorella sp. in a synthetic softwater representative of fresh surface waters in sandy-streams of tropical northern Australia. The effects of Cu and U on algal growth (cell division) rate after a 72-h exposure were determined. Intracellular and extracellular (membrane-bound) metal concentrations at the two selected pH values were also compared. Based on the 72-h minimum detectable effect concentrations (MDEC), Chlorella sp. was approximately 20-fold more sensitive to Cu (0.7 and 1.4 μg l(-1) at pH 6.5 and 5.7, respectively) than U (13 and 34 μg l(-1) at pH 6.5 and 5.7, respectively), and more sensitive than other Australian tropical freshwater organisms. The toxicity of Cu and U was highly pH-dependent. Copper concentrations required to inhibit growth (cell division) rate by 50% (72-h EC(50)) increased from 1.5 to 35 μg l(-1) as the pH decreased from 6.5 to 5.7. Similarly, the 72-h EC(50) values for U increased from 44 to 78 μg l(-1) over the same pH range. Calculation of Cu and U speciation using the geochemical model HARPHRQ, showed that differences in the concentrations of the free metal ions (Cu(2+) and UO(2)(2+)) were only minimal (<10%) between pH 5.7 and 6.5. The decreased toxicity at pH 5.7 was due to lower concentrations of cell-bound and intracellular Cu and U compared to those at pH 6.5. These results are explained in terms of the possible mechanism of competition between H(+) and the metal ion at the cell surface.     

Physiological and molecular effects associated with palladium treatment in Pseudokirchneriella subcapitata

Human activities have increased the levels of environmental palladium (Pd) worldwide. Due to the growing evidence of its toxicity, Pd pollution has become the focus of serious concern. Several studies have given an account of the increasing concentration of Pd in aquatic ecosystems. The aim of the current study is to analyze the physiological and molecular effects induced by Pd on freshwater unicellular green algae. To do this, Pseudokirchneriella subcapitata (P. subcapitata) was exposed in vitro to different concentrations (0.1, 0.25 and 0.5 mg l(-1)) of K(2)PdCl(4), a soluble salt of Pd, corresponding to 0.03, 0.075 and 0.15 mg l(-1) of Pd. The uptake and the effects on algal growth and morphology were determined. The main results are that Pd is able to induce damage in P. subcapitata at a concentration of 0.1 mg l(-1) of K(2)PdCl(4), with the damage becoming more evident at a concentration of 0.25 mg l(-1)of K(2)PdCl(4); at a concentration of 0.5 mg l(-1) of K(2)PdCl(4), cellular degeneration occurs. The main cellular target of Pd is the chloroplast, as shown by TEM and proteomic analysis. TEM analysis also showed accumulation of precipitates, probably of Pd, in the chloroplasts, although further experiments are necessary to confirm that these are Pd-precipitates. Amplified fragment length polymorphism analysis (AFLP) demonstrated that Pd, even at the lowest concentration tested, induced randomly distributed DNA changes either directly or indirectly in the algal genome and that oxidative processes were involved.     

Contrasting effects of chloride on the toxicity of silver to two green algae, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii

Short-term silver toxicity was determined for two freshwater algae, Pseudokirchneriella subcapitata and Chlamydomonas reinhardtii, in the presence and absence of chloride. Silver speciation in the exposure media was controlled and algal growth was measured over 6 h. For P. subcapitata, an alga with low Ag uptake fluxes, silver toxicity could be predicted on the basis of the free Ag+ concentration, in the presence or absence of significant complexation by chloride ions, as predicted by the biotic ligand model (BLM). For C. reinhardtii, an alga with high Ag uptake fluxes, silver toxicity was better predicted by the concentration of all labile dissolved Ag species than by free silver, a result that is consistent with diffusion through the unstirred layer surrounding the cell surface being the rate-limiting step in silver uptake. For both species, growth inhibition could be predicted on the basis of the Ag intracellular quota in the presence or absence of chloride, indicating that silver toxicity is a direct result of intracellular accumulation rather than cell surface interactions.     

Topotecan is a substrate for multidrug resistance associated protein 4

Topotecan (TPT) is a semisynthetic water-soluble derivative of camptothecin (CPT) used as second-line therapy in patients with metastatic ovarian carcinoma, small cell lung cancer, and other malignancies. However, both dose-limiting toxicity and tumor resistance hinder the clinical use of TPT. The mechanisms for resistance to TPT are not fully defined, but increased efflux of the drug by multiple drug transporters including P-glycoprotein (PgP), multidrug resistance associated protein 1 (MRP1) and breast cancer resistance protein (BCRP) from tumor cells has been highly implicated. This study aimed to investigate whether overexpression of human MRP4 rendered resistance to TPT by examining the cytotoxicity profiles using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay and cellular accumulation of TPT in HepG2 cells stably overexpressing MRP4. Two kinds of cell lines, HepG2 with insertion of an empty vector plasmid (V/HepG2), HepG2 cells stably expressing MRP4 (MRP4/HepG2), were exposed to TPT for 4 or 48 hr in the absence or presence of various MRP4 inhibitors including DL-buthionine-(S,R)-sulphoximine (BSO), diclofenac, celecoxib, or MK-571. The intracellular accumulation of TPT and paclitaxel (a PgP substrate) by V/HepG2 and MRP4/HepG2 cells was determined by incubation of TPT with the cells and the amounts of the drug in cells were determined by validated HPLC methods. The study demonstrated that MRP4 conferred a 12.03- and 6.86-fold resistance to TPT in the 4- and 48-hr drug-exposure MTT assay, respectively. BSO, MK-571, celecoxib, or diclofenac sensitised MRP4/HepG2 cells to TPT cytotoxicity and partially reversed MRP4-mediated resistance to TPT. In addition, the accumulation of TPT was significantly reduced in MRP4/HepG2 cells compared to V/HepG2 cells, and one-binding site model was found the best fit for the MRP4-mediated efflux of TPT, with an estimated K(m) of 1.66 microM and V(max) of 0.341 ng/min/106 cells. Preincubation of MRP4/HepG2 cells with BSO (200 microM) for 24 hr, celecoxib (50 microM), or MK-571 (100 microM) for 2 hr significantly increased the accumulation of TPT over 10 min in MRP4/HepG2 cells by 28.0%, 37.3% and 32.5% (P < 0.05), respectively. By contrast, there was no significant difference in intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells over 120 min. MRP4 also rendered resistance to adefovir dipivoxil (bis-POM-PMEA) and methotrexate, two reported MRP4 substrates. MRP4 did not exhibit any significant resistance to other model drugs including vinblastine, vincristine, etoposide, carboplatin, cyclosporine and paclitaxel in both long (48 hr) and short (4 hr) drug-exposure MTT assays. These findings indicate that MRP4 confers resistance to TPT and TPT is the substrate for MRP4. Further studies are needed to explore the role of MRP4 in resistance to, toxicity and pharmacokinetics of TPT in cancer patients.     

Effects of microcystin and complex cyanobacterial samples on the growth and oxidative stress parameters in green alga Pseudokirchneriella subcapitata and comparison with the model oxidative stressor—herbicide paraquat

Oxidative stress is one of the biochemical mechanisms involved in toxicity of cyanobacterial toxins microcystins (MC), but its role in the effects of complex water blooms is elusive. The aim of this study was to investigate effects of pure MCs and different complex mixtures of cyanobacterial metabolites on the growth and biochemical markers of oxidative stress and detoxification in green alga Pseudokirchneriella subcapitata. Pure MCs at high concentrations (300 μg/L) had no effects on the growth of P. subcapitata (up to 10 day exposures) but stimulated activity of glutathione reductase (GR) after short 3 and 24 h exposures. Other biomarkers (levels of glutathione, GSH, and activities of glutathione‐S‐transferase, GST, and glutathione peroxidase, GPx) were not affected by pure MCs). Crude extract of the laboratory culture of cyanobacteria Microcystis aeruginosa (containing 300 μg/L of MCs) had no effects on algal growth or any of the biomarkers. Weak growth stimulations after 4–7 days were observed after exposures to the growth‐spent medium of the M. aeruginosa culture, which also inhibited activities of GST after prolonged exposures. Other investigated parameters (reduced GSH and activity of GPx) were not affected by any of the cyanobacterial samples. The results were compared with effects of model oxidative stressor herbicide paraquat, which exhibited variable effects on both algal growth and biomarkers (decrease in reduced GSH, stimulations of GR). Taken together, although pure MCs induce oxidative stress in green alga, the effects of cyanobacterial mixtures, which are more relevant to the natural situation, are more complex and they differ from the pure toxin. High variability in the biochemical responses to the oxidative stress makes the interpretation of results complicated, which limits the use of these biomarkers as early warnings of toxicity under natural conditions. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

拓扑替康对宫颈癌HeLa细胞的抑制及放疗增敏作用

目的研究拓扑替康(TPT)对宫颈癌HeLa细胞的抑制和放疗增敏作用。方法噻唑蓝法(MTT)和流式细胞仪法(FCM)检测不同浓度TPT在不同作用时间下对HeLa细胞的抑制作用;细胞克隆形成法进一步检测TPT作用24h和48h后对HeLa细胞抑制及放疗增敏作用,应用单击多靶模型拟合细胞存活曲线并计算放疗增敏比。结果TPT对宫颈癌HeLa细胞的抑制作用呈时间剂量依赖性,作用浓度越大、作用时间越长,对肿瘤细胞的抑制程度越大(P<0.05)。TPT作用24h时的半数致死浓度(IC50)为8μg/mL,显著低于作用48h和72h时IC50(P<0.05)。FCM检测TPT联合放射线辐射对HeLa细胞的抑制率为68.2%,明显高于单化疗组(45.7%)和单放射线辐射组(14.4%)(P<0.05)。细胞克隆形成法表明TPT作用24h和48h后联合不同剂量放射线,对HeLa细胞的杀伤作用明显增强,提示TPT具有放疗增敏作用。应用单击多靶模型拟合生存曲线得到24h和48h放疗增敏比为1.167和1.344。结论TPT对宫颈癌HeLa细胞具有抑制和放疗增敏作用,呈时间剂量依赖性,其机制可能与细胞放射线损伤修复功能抑制有关。     

Endogenous 4-hydroxy-2-nonenal in microalga Chlorella kessleri acts as a bioactive indicator of pollution with common herbicides and growth regulating factor of hormesis

Oxidative stress, i.e. excessive production of reactive oxygen species (ROS), leads to lipid peroxidation and to formation of reactive aldehydes (e.g. 4-hydroxy-2-nonenal; HNE), which act as second messengers of free radicals. It was previously shown that herbicides can induce ROS production in algal cells. In the current paper, the unicellular green microalga Chlorella kessleri was used to study the effect of two herbicides (S-metolachlor and terbuthylazine) and hydrogen peroxide (H(2)O(2)) on oxidative stress induction, HNE formation, chlorophyll content and the cell growth. Production of HNE was detected in this study for the first time in the cells of unicellular green algae using the antibody specific for the HNE-histidine adducts revealing the HNE-histidine adducts even in untreated, control C. kessleri. Exposure of algal cells to herbicides and H(2)O(2) increased the ROS production, modifying production of HNE. Namely, 4h upon treatment the levels of HNE-histidine conjugates were below controls. However, their amount increased afterwards. The increase of HNE levels in algae was followed by their increased growth rate, as was previously described for human carcinoma cells. Hence, changes in the cellular HNE content upon herbicide treatment inducing lipid oxidative stress and alterations in cellular growth rate of C. kessleri resemble adaptation of malignant cells to the HNE treatment. Therefore, as an addition to the standard toxicity tests, the evaluation of HNE-protein adducts in C. kessleri might indicate environmental pollution with lipid peroxidation-inducing herbicides. Finally, C. kessleri might be a convenient experimental model to further study cellular hormetic adaptation to oxidative stress-derived aldehydes.     

维拉帕米对抗阿霉素的中国仓鼠卵巢上皮细胞抗药性的逆转作用

非细胞毒性剂量的维拉帕米(Ver)3μg/ml,增强阿霉素(ADM)对抗阿霉素的中国仓鼠卵巢上皮细胞(RC₁)的生长抑制达10倍。用集落形成法测定表明:Ver 3μg/ml能降低RC₁的IC₅₀值,即从1.2μg/ml降至0.08μg/ml,逆转抗药性达15倍。由于Ver增强ADM在RC₁细胞的积累,从而增强ADM对RC₁细胞的毒性,用流式细胞光度计,研究Ver逆转抗药性机制,发现Ver与ADM合用,能阻断RC₁细胞在G₂+M期。     

Kinetics of copper accumulation in Lessonia nigrescens (Phaeophyceae) under conditions of environmental oxidative stress

Juvenile individuals of the brown kelp Lessonia nigrescens were exposed to a coastal environment chronically impacted by copper mine wastes and currently displaying more than 250 nM of total dissolved copper. The kinetic of copper accumulation in the intra and extracellular compartments was determined and correlated to the oxidative burst resulting from copper-mediated oxidative stress. Accumulation involved an initial adsorption onto the outer cell wall followed by a slower uptake into the cells. A linear pattern of copper uptake over time was found during the first 52 h of exposure, and a steady state was reached at 76 h. The resulting oxidative stress was found to be inefficiently attenuated, and the intracellular level of copper remained sufficiently high to determine a persistently higher than normal level of reactive oxygen species (ROS). Thus, our results strongly suggest that, in L. nigrescens, copper needs to reach an intracellular threshold before oxidative burst develops. Furthermore, it was found that the high ROS levels generated by copper accumulation within the cells persists after the oxidative burst has ceased, suggesting a limited capacity of the algal tissue to buffer the increases of ROS caused by the environmental copper levels.     

The influence of iron on copper toxicity to the marine diatom, Nitzschia closterium (ehrenberg) W. Smith

The effect of iron on the toxicity of copper (0–60 μg Cu l^?1) to the marine diatom, Nitzschia closterium (Ehrenberg) W. Smith was studied using unsupplemented sea water for growth rate experiments. Intracellular and extracellular iron and copper concentrations were determined by inductively coupled plasma emission spectrometry and anodic stripping voltammetry.Cells cultured under normal f medium iron levels (790 μg Fe·l^?1) were more tolerant to copper than cells cultured in iron deficient medium (79 μg Fe·l^?1 and 7.9 μg Fe·l^?1). Lower extracellular iron and higher intracellular copper concentrations were found in cells grown previously in iron deficient medium compared to cells grown in normal iron medium. Iron, however, had no effect on the toxicity of the lipid soluble copper complex, copper-oxine.It is proposed that copper toxicity is reduced by colloidal ferric hydroxide, carried over in the assay inoculum, which binds to the diatom cell membrane, absorbs copper and prevents copper penetration into the cell.     

Apparent toxicity resulting from the sequestering of nutrient trace metals during standard Selenastrum capricornutum toxicity tests

The water accommodated fractions (WAFs) of aqueous mixtures of three lubricant additives showed significant apparent toxicity to the freshwater alga, Selenastrum capricornutum, and experiments were conducted to investigate the hypothesis that toxicity resulted from the removal of one or more essential nutrients from the test medium by the lubricant additives. Algal growth effects were noted at ashless dispersant A concentrations as low as 0.5 x mg l(-1) and growth was completely inhibited at 100 mg x l(-1). Algal cells transferred from the 100 mg x l(-1) WAF of ashless dispersant A to fresh medium at the end of a standard 96-h toxicity test resumed growing at a rate similar to growth in undosed algal medium, indicating that the effect was algistatic rather than algicidal. Fortifying the iron (Fe) and disodium ethylenediaminetetraacetic acid (EDTA) concentrations of the WAF with 200% of the concentrations used in the formulation of algal medium after 24 h of exposure caused a resumption of algal growth at a rate comparable to the control growth, and a resulting EL50 value above 100 mg x l(-1). Similar effects were observed when the two other lubricant additives were tested at WAF concentrations that completely inhibited algal growth during standard toxicity tests: fortification of a 50 mg x l(-1) WAF of ZnDTP with 1000% of the Fe and EDTA used in the formulation of algal medium caused a resumption of growth at a rate statistically identical to the control growth, and the fortification of a 2800 mg x l(-1) WAF of ashless dispersant B with 700% of the entire complement of nutrients used in the formulation of algal medium caused a resumption of growth at a rate comparable to the control. The indirect toxic effect of these lubricant additives to algae results from the sequestration of one or more nutrient metals essential for algal growth. Standard algal toxicity tests with these lubricant additives may, therefore, have little environmental relevance because the complex chemistry of natural waters differs greatly from the nutrient limited algal medium, and the sequestration effect observed in these static tests could be absent under real world conditions.     

Relationships between acid-soluble thiol peptides and accumulated Pb in the green alga Stichococcus bacillaris

Stichococcus bacillaris, an ubiquitous green microalga accumulated inorganic lead (Pb) from aqueous solutions extra- and intracellularly. In response to Pb uptake acid-soluble thiol peptides (glutathione - GSH and phytochelatins - PC) were synthesized. The proportion of the intracellular Pb uptake by algal cells was low and comprised only 3-6% of the total metal sorption. The intracellular uptake was dependent on external Pb concentration, time of metal exposure and cell metabolism. Pb accumulation in alga was determined by means of 210Pb radiometry. Reduced GSH and PC were determined in algal cells using HPLC with the post-column derivatization with Ellman's reagent. Within the studied concentration range 0.1-20 μM, inorganic lead caused a significant production of induced thiol peptides: PC (n=2-4) and some other unidentified oligopeptides, probably (GluCys)n. The time of appearance and the concentration of individual oligomers of phytochelatins were dependent on the external Pb concentration and time of metal exposure. In algal cells exposed to Pb, significant changes in the GSH level accompanying the formation of the induced thiol peptides were also observed. The GSH level decreased in the cells exposed to the lower (up to 10 μM) studied Pb concentrations or increased in the cells treated with higher (20 μM) Pb concentrations. The thiol groups originated from induced peptides (mainly phytochelatins) followed a stoichiometric relationship 2:1 to the intracellular Pb amounts, however, only at the lowest studied external concentration (0.1 μM). At higher concentrations (up to 2.5 μM), intracellular Pb concentration was equal or even exceeded (at Pb>2.5 μM) two to three times the level of induced thiols. S. bacillaris accumulated intracellularly by 46% more Pb in light than in dark and the level of induced thiol peptides was significantly higher in the cells exposed to Pb under illumination. The rapid formation of these peptides in S. bacillaris in response to Pb, and their elimination (by about 90%) when algae were placed into the Pb-free solution reveal a tight regulation of GSH and phytochelatin pools in the algal cells exposed to toxic metals. The obtained results suggest that both PCs and GSH are the primary line of defence against the Pb toxicity. Additionally, the induced thiol peptides in S. bacillaris could be a good indicator of intracellular Pb availability and stress at the metal concentrations found in polluted fresh waters.     

Oxidative damage to biological macromolecules in human bone marrow mesenchymal stromal cells labeled with various types of iron oxide nanoparticles

The biological effects of several superparamagnetic iron oxide nanoparticles (SPIONs) varying in their surface coating were tested using human bone marrow mesenchymal stromal cells from two donors - hBMSCs-1 and hBMSCs-2. The measurements were performed at two intervals - after 72 h exposure to the nanoparticles and after an additional 72 h cell growth without nanoparticles. The dose of SPIONs used (15.4 μg Fe/ml) was selected as being sufficient for in vivo cell tracking using magnetic resonance imaging (MRI). Concerning cell viability and cell death, only the hBMSCs-2 seemed to be sensitive to the action of SPIONs. However, an increase of oxidative injury to lipids, proteins and DNA as a consequence of exposure to SPIONs was detected in cells from both donors. Particularly the levels of lipid peroxidation were high and increased further with time, regardless of the type of nanoparticle. Lowering intracellular label concentrations and authenticating oxidative stress levels using in vivo experiments are required to ensure the safety of SPIONs for biomedical applications.     

Cadmium toxicity to two marine phytoplankton under different nutrient conditions

Cd accumulation and toxicity in two marine phytoplankton (diatom Thalassiosira weissflogii and dinoflagellate Prorocentrum minimum) under different nutrient conditions (nutrient-enriched, N- and P-starved conditions) were examined in this study. Strong interactions between the nutrients and Cd uptake by the two algal species were found. Cd accumulation as well as N and P starvation themselves inhibited the assimilation of N, P, and Si by the phytoplankton. Conversely, N starvation strongly inhibited Cd accumulation but no influence was observed under P starvation. However, the Cd accumulation difference between nutrient-enriched and N-starved cells was smaller when [Cd(2+)] was increased in the medium, indicating that net Cd accumulation was less dependent on the N-containing ligands at high-Cd levels. As for the subcellular distribution of the accumulated Cd, most was distributed in the insoluble fraction of T. weissflogii while it was evenly distributed in the soluble and insoluble fractions of P. minimum at low-Cd levels. A small percentage of cellular Cd (<15%) was adsorbed on the cell surface for both algae at the lowest [Cd(2+)], which increased when the [Cd(2+)] increased. Cd toxicity in phytoplankton was quantified as depression of growth and maximal photosynthetic system II quantum yield, and was correlated with the [Cd(2+)], intracellular Cd concentration, and Cd concentrations in the cell-surface-adsorbed, soluble, and insoluble fractions. According to the estimated median inhibition concentration (IC50) based on the different types of Cd concentration, the toxicity difference among the different nutrient-conditioned cells was the smallest when the Cd concentration in the soluble fraction was used, suggesting that it may be the best predictor of Cd toxicity under different nutrient conditions.     

Reversibility of copper-thiol binding in Nitzschia closterium and Chlorella pyrenoidosa

The effect of ionic copper and copper complexes on intracellular thiols in the marine diatom Nitzschia closterium (Ehrenberg) W. Smith, and the green alga Chlorella pyrenoidosa Chick was studied using the lipid-soluble thiol reagent, dithiodipyridine. Treatment of cells with copper ions significantly reduced the concentration of intracellular thiol groups, whereas copper complexes and H₂O₂ had no effect.To test the reversibility of intracellular copper-thiol binding in N. closterium and C. pyrenoidosa, cells exposed to copper (60–200 μg Cu·1⁻¹) were treated with various sulfhydryl compounds and chelating agents, with and without removal of copper from solution. Cysteine, glutathione (GSH), EDTA and bathocuproine disulfonate were most effective in reversing copper toxicity, whereas oxidized glutathione, cystine and glycine had no effect. It is proposed that copper (II) adsorbed to the algae is reduced to copper (I) by -SH groups, and bound as a Cu(I)-S-complex. Sulfhydryl compounds can reverse toxicity through thiol-disulfide interchange reactions, allowing algal growth to recommence, and powerful copper-chelating agents produce the same effect by withdrawing copper from the cell.     

Inhibitory effects of extracts from the marine alga Caulerpa taxifolia and of toxin from Caulerpa racemosa on multixenobiotic resistance in the marine sponge Geodia cydonium

The invasive growth of the introduced green alga Caulerpa taxifolia, already affecting the richness and diversity of the littoral ecosystems, has become a major ecological problem in the Mediterranean Sea. Previously, we demonstrated that the water pollutant tributyltin induces apoptosis in tissue of the marine sponge Geodia cydonium at concentrations of 3 μM and higher. Here we show that exposure of G. cydonium to low (non-toxic) concentrations of Caulerpa extract or purified caulerpin (10 μg/ml) together with low doses of tributyltin (1 μM; non-toxic), results in a strong apoptotic effect. Evidence is presented that the enhancement of toxicity of tributyltin by Caulerpa extract is at least partially caused by inhibition of the multixenobiotic resistance (MXR) pump by the algal toxin. Caulerpa extract, as well as caulerpin, strongly enhance the accumulation of the test substrate of MXR, rhodamine B, in the gills of the mussel Dreissena polymorpha, used as a model system for testing MXR-inhibiting potential.     

Scutellarin attenuates oxidative glutamate toxicity in PC12 cells

The present study investigated the protective effects of the antioxidant scutellarin against oxidative toxicity induced by glutamate in PC12 cells. Vitamin E, a classical antioxidant was employed as a comparative agent. Incubation of PC12 cells with 10 mM glutamate resulted in significant cytotoxity as evaluated by the MTT and lactate dehydrogenase (LDH) assays, decreases of GSSG reductase activity, disturbance of the cell redox state as indicated by the GSH/GSSG ratio, and accumulation of intracellular reactive oxygen species (ROS) and lipid peroxidation products. Scutellarin at 0.1, 1 and 10 microM significantly protected against the cytoxicity and production of ROS and lipid peroxidation induced by glutamate. Scutellarin did not prevent the reduction of cellular GSH levels, but it up-regulated GSSG reductase activity, thus preventing an increase in cellular GSSG levels, and concomitantly improved the cell redox status. Our data also show that the protective effects of scutellarin against glutamate-induced oxidative toxicity are more potent than that of vitamin E. These results demonstrate that scutellarin can protect PC12 cells from oxidative glutamate toxicity by scavenging ROS, inhibiting lipid peroxidation and improving the cell redox status, and may reduce the cellular damage in pathological conditions associated with excessive glutamate release.     

Evaluation of pulmonary toxicity of benzalkonium chloride and triethylene glycol mixtures using in vitro and in vivo systems

Benzalkonium chloride (BAC) is a widely used disinfectant/preservative, and respiratory exposure to this compound has been reported to be highly toxic. Spray‐form household products have been known to contain BAC together with triethylene glycol (TEG) in their solutions. The purpose of this study was to estimate the toxicity of BAC and TEG mixtures to pulmonary organs using in vitro and in vivo experiments. Human alveolar epithelial (A549) cells incubated with BAC (1‐10 μg/mL) for 24 hours showed significant cytotoxicity, while TEG (up to 1000 μg/mL) did not affect cell viability. However, TEG in combination with BAC aggravated cell damage and inhibited colony formation as compared to BAC alone. TEG also exacerbated BAC‐promoted production of reactive oxygen species (ROS) and reduction of glutathione (GSH) level in A549 cells. However, pretreatment of the cells with N‐acetylcysteine (NAC) alleviated the cytotoxicity, indicating oxidative stress could be a mechanism of the toxicity. Quantification of intracellular BAC by LC/MS/MS showed that cellular distribution/absorption of BAC was enhanced in A549 cells when it was exposed together with TEG. Intratracheal instillation of BAC (400 μg/kg) in rats was toxic to the pulmonary tissues while that of TEG (up to 1000 μg/kg) did not show any harmful effect. A combination of nontoxic doses of BAC (200 μg/kg) and TEG (1000 μg/kg) promoted significant lung injury in rats, as shown by increased protein content and lactate dehydrogenase (LDH) activity in bronchoalveolar lavage fluids (BALF). Moreover, BAC/TEG mixture recruited inflammatory cells, polymorphonuclear leukocytes (PMNs), in terminal bronchioles and elevated cytokine levels, tumor necrosis factor α (TNF‐α), and interleukin 6 (IL‐6) in BALF. These results suggest that TEG can potentiate BAC‐induced pulmonary toxicity and inflammation, and thus respiratory exposure to the air mist from spray‐form products containing this chemical combination is potentially harmful to humans.     

The effect of water hardness on the toxicity of uranium to a tropical freshwater alga Chlorella sp

Uranium (U) derived from mining activities is of potential ecotoxicological concern to freshwater biota in tropical northern Australia. Few data are available on the effects of water hardness (Ca and/or Mg), which is elevated in U mine wastewaters, on the toxicity and bioavailability of U to freshwater biota, particularly algae. This study determined the effect of water hardness (8, 40, 100 and 400 mg CaCO(3) x l(-1), added as calcium (Ca) and magnesium (Mg) sulphate) on the toxicity (72 h growth rate inhibition) of U to the unicellular green alga, Chlorella sp., in synthetic freshwater, at constant pH (7.0) and alkalinity (8 mg CaCO(3) x l(-1)), similar in chemical composition to sandy coastal streams in tropical northern Australia. A 50-fold increase in water hardness resulted in a 5-fold decrease (P0.05) differences in the predicted speciation (% distribution) of U amongst the four water hardness levels. The reduction in U toxicity with increasing water hardness was most likely due to competition between U and Ca and/or Mg for binding sites on the algal cell surface. The minimum detectable effect concentrations of U were approximately 3 and 24 times higher (at 8 and 400 mg CaCO(3)x l(-1) hardness, respectively) than the national interim U guideline value (0.5 micro g x l(-1)) for protecting aquatic ecosystems. Overall, the results reinforce the need for a more flexible U guideline based on a hardness-dependent algorithm, which may allow environmental managers to relax the national guideline for U on a site-specific basis.     

Interactions of copper and manganese: A mechanism by which manganese alleviates copper toxicity to the marine diatom, Nitzschia closterium (Ehrenberg) W. Smith

The mechanism by which manganese (4.2 and 42 μg Mn·1^?1) ameliorates the toxicity of copper (0–60 μg Cu·1^?1) to the marine diatom, Nitzschia closterium (Ehrenberg) W. Smith, was investigated using unsupplemented sea water for growth rate experiments. Speciation of manganese associated with the cellls, intracellular and extracellular manganese and copper concentrations, and competitive binding between copper and manganese were studied using ultrafiltration, anodic stripping voltammetry and radiochemical techniques.Cells cultured before the sea-water assay and in the absence of manganese accumulated high intracellular copper, and their growth rate was more sensitive to copper than cells cultured in the presence of manganese. Manganese associated with the cells was present as manganese (II) and/or (III) hydroxides, rather than as the fully oxidized MnO₂. It is proposed that manganese (III) hydroxide, like iron (III) hydroxide and cobalt (III) hydroxide, adsorbs copper very effectively on the membrane surface and prevents its penetration into the cell. Competitive interactions between manganese and copper occurred at the cell surface, but copper had no effect on intracellular manganese. Only 4 μg Mn·1^?1 was required to alleviate copper toxicity, compared to 790 μg Fe·1^?1. In the presence of algae, copper ions had a greater affinity for manganese in sea water than iron at similar concentrations, which may partially account for the relative effectiveness of manganese as a protective agent. In addition, manganese can scavenge the toxic superoxide radical (O₂^?), catalyzing its dismutation to H₂O₂ and O₂.Manganese was unable to reverse copper toxicity, nor did it inhibit the toxicity of lipid-soluble copper complexes, such as copper oxinate, to N. closterium.