Immunostimulatory effects of the intracellular polysaccharides isolated from liquid culture of Ophiocordyceps sinensis (Ascomycetes) on RAW264.7 cells via the MAPK and PI3K/Akt signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Chinese Cordyceps (DongChong XiaCao), a parasitic complex of a fungus Ophiocordyceps sinensis and a caterpillar, is a traditional Chinese medicine. Polysaccharides extracted from O. sinensis have immunomodulatory effects on macrophages. However, the mechanism of polysaccharides on macrophage and the composition of polysaccharides are not known. AIM OF STUDY: We aimed to investigate composition and structure of the intracellular polysaccharides from O. sinensis mycelia (designed as OSP), and evaluate its the immunomodulatory effect on macrophages and its underlying mechanism. MATERIALS AND METHODS: We performed a liquid-state fermentation of O. sinensis to produce mycelia. The DEAE-Sephadex-A25 cellulose column and Sephadex-G100 gel column chromatography were employed to purify and character the intracellular OSP. Macrophages RAW264.7 cells were employed to evaluate OSP's immunomodulatory activity and the possible mechanism responsible for the activation of macrophages in vitro. RESULTS: The average molecular weight of OSP was distributed at 27,972 Da, OSP was composed of xylose, mannose, glucose, and galactose with the ratio of 2.9 : 6.6 : 166 : 2.6, with a trace amount of fucose, arabinose and rhamnose. The phagocytosis of RAW264.7 cells was improved significantly and remarkable changes were observed in the morphology with OSP-treated cells. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis demonstrated that OSP had an ability to regulate the mRNA expression of pro-inflammatory and anti-inflammatory cytokines, and to induce the mRNA expression level of iNOS in a concentration dependent manner in RAW264.7 cells. Western blotting analysis showed that the regulation of NO and cytokines was mediated through mitogen-activated protein kinase (MAPK) and PI3K/Akt signaling pathways. CONCLUSION: This study demonstrated that OSP was with a capacity to activate macrophage cells RAW264.7 for an improvement of immunomodulation activities, which was through regulation of inflammatory mediators via MAPK and PI3K/Akt signaling pathways.

Intracellular Photothermal Delivery for Suspension Cells Using Sharp Nanoscale Tips in Microwells.

Efficient intracellular delivery of biomolecules into cells that grow in suspension is of great interest for biomedical research, such as for applications in cancer immunotherapy. Although tremendous effort has been expended, it remains challenging for existing transfer platforms to deliver materials efficiently into suspension cells. Here, we demonstrate a high-efficiency photothermal delivery approach for suspension cells using sharp nanoscale metal-coated tips positioned at the edge of microwells, which provide controllable membrane disruption for each cell in an array. Self-aligned microfabrication generates a uniform microwell array with three-dimensional nanoscale metallic sharp tip structures. Suspension cells self-position by gravity within each microwell in direct contact with eight sharp tips, where laser-induced cavitation bubbles generate transient pores in the cell membrane to facilitate intracellular delivery of extracellular cargo. A range of cargo sizes were tested on this platform using Ramos suspension B cells with an efficiency of >84% for Calcein green (0.6 kDa) and >45% for FITC-dextran (2000 kDa), with retained viability of >96% and a throughput of >100 000 cells delivered per minute. The bacterial enzyme ß-lactamase (29 kDa) was delivered into Ramos B cells and retained its biological activity, whereas a green fluorescence protein expression plasmid was delivered into Ramos B cells with a transfection efficiency of >58%, and a viability of >89% achieved.

Magnetic Relaxation of Intracellular Magnetic Nanoparticles for Hyperthermia.

Magnetic nanoparticles have been studied extensively for biomedical applications over the past decades. One of the promising applications of magnetic nanoparticles is hyperthermia, which refers to thermal treatment for cancer. To achieve adequate heat at target sites, it is essential to develop magnetic nanoparticles with high heating efficiency and to optimize external magnetic fields. Here, we discuss the heating mechanism of magnetic nanoparticles, the influence of the intracellular environment on magnetic behavior and heat generation, and recent advances in methods of heating efficiency assessment.

Exploring naturally occurring ivy nanoparticles as an alternative biomaterial.

Arabinoglactan protein (AGP)-rich nanoparticles obtained from the sticky exudates of Hedera helix (English ivy), have shown promising potential to be used in nanomedicine owing to their excellent aqueous solubility, low intrinsic viscosity, biocompatibility, and biodegradability. In this study, the feasibilities of utilizing ivy nanoparticles (INPs) as nano-carriers for delivering chemotherapeutic drugs in cancer therapy and as nano-fillers to develop novel scaffolds for tissue engineering in regenerative medicine are evaluated. Via electrostatic and hydrophobic interactions, pH-responsive nanoconjugates are formed between the INPs and the doxorubicin (DOX) with an entrapment ratio of 77.9±3.9%. While the INPs show minimal cytotoxicity, the formed INP-DOX conjugates exhibit substantially stronger cytotoxic activity than free DOX against multiple cancer cell lines, suggesting a synergistic effect is established upon conjugation. The anti-cancer effects of the INP-DOX conjugates are further evaluated via in vivo xenograft assays by subcutaneously implanting DOX resistant cell line, SW620/Ad-300, into nude mice. The tumor volumes in mice treated with the INP-DOX conjugates are significantly less than those of the mice treated with free DOX. In addition, the INPs are further exploited as nano-fillers to develop fibrous scaffolds with collagen, via mimicking the porous matrix where the INPs are embedded under natural condition. Enhanced adhesion of smooth muscle cells (SMCs) and accelerated proliferation of mouse aortic SMCs are observed in this newly constructed scaffold. Overall, the results obtained from the present study suggest great potential of the INPs to be used as biocompatible nanomaterials in nanomedicine. The AGP-rich INP renders a glycoprotein architecture that is amenable for modification according to the functional designs, capable of being developed as versatile nanomaterials for extensive biomedical applications. STATEMENT OF SIGNIFICANCE: Naturally occurring organic nanomaterials have drawn increasing interest for their potential biomedical applications in recent years. In this study, a new type of naturally occurring nanoparticles obtained from the sticky exudates on the adventitious roots of English ivy (H. helix), was explored for its potential biomedical application. In particular, the feasibilities of utilizing ivy nanoparticles (INPs) as nano-carriers for delivering chemotherapeutic drugs in cancer therapy and as nano-fillers to develop novel scaffolds for tissue engineering in regenerative medicine were evaluated both in vitro and in vivo. Overall, the results obtained from the present study suggest the great potential of the INPs to be used as biocompatible nanomaterials in nanomedicine. This study may open a totally new frontier for exploring the biomedical application of naturally occurring nanomaterials.

Intracellular accumulation dynamics and fate of zinc ions in alveolar epithelial cells exposed to airborne ZnO nanoparticles at the air-liquid interface.

Airborne nanoparticles (NPs) that enter the respiratory tract are likely to reach the alveolar region. Accumulating observations support a role for zinc oxide (ZnO) NP dissolution in toxicity, but the majority of in-vitro studies were conducted in cells exposed to NPs in growth media, where large doses of dissolved ions are shed into the exposure solution. To determine the precise intracellular accumulation dynamics and fate of zinc ions (Zn(2+)) shed by airborne NPs in the cellular environment, we exposed alveolar epithelial cells to aerosolized NPs at the air-liquid interface (ALI). Using a fluorescent indicator for Zn(2+), together with organelle-specific fluorescent proteins, we quantified Zn(2+) in single cells and organelles over time. We found that at the ALI, intracellular Zn(2+) values peaked 3 h post exposure and decayed to normal values by 12 h, while in submerged cultures, intracellular Zn(2+) values continued to increase over time. The lowest toxic NP dose at the ALI generated peak intracellular Zn(2+) values that were nearly three-folds lower than the peak values generated by the lowest toxic dose of NPs in submerged cultures, and eight-folds lower than the peak values generated by the lowest toxic dose of ZnSO4 or Zn(2+). At the ALI, the majority of intracellular Zn(2+) was found in endosomes and lysosomes as early as 1 h post exposure. In contrast, the majority of intracellular Zn(2+) following exposures to ZnSO4 was found in other larger vesicles, with less than 10% in endosomes and lysosomes. Together, our observations indicate that low but critical levels of intracellular Zn(2+) have to be reached, concentrated specifically in endosomes and lysosomes, for toxicity to occur, and point to the focal dissolution of the NPs in the cellular environment and the accumulation of the ions specifically in endosomes and lysosomes as the processes underlying the potent toxicity of airborne ZnO NPs.

Effect of intracellular P content on phosphate removal in Scenedesmus sp. Experimental study and kinetic expression.

The present work determines the effect of phosphorus content on phosphate uptake rate in a mixed culture of Chlorophyceae in which the genus Scenedesmus dominates. Phosphate uptake rate was determined in eighteen laboratory batch experiments, with samples taken from a progressively more P-starved culture in which a minimum P content of 0.11% (w/w) was achieved. The results obtained showed that the higher the internal biomass P content, the lower the phosphate removal rate. The highest specific phosphate removal rate was 6.5mgPO4-PgTSS(-1)h(-1). Microalgae with a P content around 1% (w/w) attained 10% of this highest removal rate, whereas those with a P content of 0.6% (w/w) presented 50% of the maximum removal rate. Different kinetic expressions were used to reproduce the experimental data. Best simulation results for the phosphate uptake process were obtained combining Steele equation and Hill function to represent the effect of light and intracellular phosphorus content, respectively.

Gracilaria lemaneiformis polysaccharide as integrin-targeting surface decorator of selenium nanoparticles to achieve enhanced anticancer efficacy.

The poor permeability of glioma parenchyma represents a major limit for antiglioblastoma drug delivery. Gracilaria lemaneiformis polysaccharide (GLP), which has a high binding affinity to αvß3 integrin overexpressed in glioma cells, was employed in the present study to functionalize selenium nanoparticles (SeNPs) to achieve antiglioblastoma efficacy. GLP-SeNPs showed satisfactory size distribution, high stability, and selectivity between cancer and normal cells. In U87 glioma cell membrane, which has a high integrin expression level, GLP-SeNPs exhibited significantly higher cellular uptake than unmodified SeNPs. As expected, U87 cells exhibited a greater uptake of GLP-SeNPs than C6 cells with low integrin expression level. Furthermore, the internalization of GLP-SeNPs was inhibited by cyclo-(Arg-Gly-Asp-Phe-Lys) peptides, suggesting that cellular uptake into U87 cells and C6 cells occurred via αvß3 integrin-mediated endocytosis. For U87 cells, the cytotoxicity of SeNPs decorated by GLP was enhanced significantly because of the induction of various apoptosis signaling pathways. Internalized GLP-SeNPs triggered intracellular reactive oxygen species downregulation. Therefore, p53, MAPKs, and AKT pathways were activated to advance cell apoptosis. These findings suggest that surface decoration of nanomaterials with GLP could be an efficient strategy for design and preparation of glioblastoma targeting nanodrugs.

Effects of 2,3-dimercaptosuccinic acid-coated Fe3O4 nanoparticles on genes in two mouse cell lines.

To evaluate the effects of iron oxide nanoparticles on genes from different cell lines in this study, mouse macrophage RAW264.7 and hepatocyte Hepa1-6 cell lines were treated with DMSA-coated Fe3O4 nanoparticles. Two doses were used, 50 microg/mL and 100 microg/mL, respectively, for 24 hr and gene expression profile was detected by DNA microarrays. The gene expression patterns for the two cell lines greatly differed from each other when compared, revealing distinct cell-specific effects of the nanoparticles on the genes. It was found that the nanoparticles significantly influenced expression of genes in the RAW264.7 cells, showing complete difference from those of Hepa1-6 cells. More genes were downregulated in the RAW264.7 cells by two doses of the nanoparticles but up-regulated in the Hepa1-6 cells by two doses of the same nanoparticles. Moreover, the increase of nanoparticle dose greatly decreased up-regulation of genes but increased down-regulation of genes in the RAW264.7 cells. The influence of nanoparticles did not result in similar effect in the Hepa1-6 cells. Apart from the difference in gene expression patterns in the two cell lines, there were eight and seven genes which were consistently down-regulated and up-regulated by low-dose and high-dose of nanoparticles in the two cell lines, respectively, revealing the common effects of the nanoparticles on the genes in the two cell lines. These common effects on genes were caused by homeostatic processes, influenced by down-regulation of genes and immune responses, and also cell death, influenced by up-regulation of genes. The data from this study has shed new insights into the potential in vivo nanotoxicolgy of the DMSA-coated Fe3O4 nanoparticles.

Thermally responsive nanoparticle-encapsulated curcumin and its combination with mild hyperthermia for enhanced cancer cell destruction.

In this study, thermally responsive polymeric nanoparticle-encapsulated curcumin (nCCM) was prepared and characterized. The nCCM is ≈ 22 and 300 nm in diameter at 37 and 22 °C, respectively. The smaller size of the nCCM at 37 °C was found to significantly facilitate its uptake in vitro by human prostate adenocarcinoma PC-3 cancer cells. However, the intracellular nCCM decreases rapidly (rather than plateaus) after reaching its peak at ≈ 1.5 h during a 3-day incubation of the PC-3 cells with nCCM. Moreover, a mild hyperthermia (with negligible cytotoxicity alone) at 43 °C applied between 1 and 1.5 h during the 3-day incubation not only increases the peak uptake but also alters intracellular distribution of nCCM (facilitating its delivery into cell nuclei), which helps to retain a significantly much higher level of intracellular curcumin. These effects of mild hyperthermia could be due in part to the thermal responsiveness of the nCCM: they are more positively charged at 43 °C and can be more easily attracted to the negatively charged nuclear membrane to enter nuclei as a result of electrostatic interaction. Ultimately, a combination of the thermally responsive nCCM and mild hyperthermia significantly enhances the anticancer capability of nCCM, resulting in a more than 7-fold decrease in its inhibitory concentration to reduce cell viability to 50% (IC50). Further mechanistic studies suggest injury pathways associated with heat shock proteins 27 and 70 should contribute to the enhanced cancer cell destruction by inducing cell apoptosis and necrosis. Overall, this study demonstrates the potential of combining mild hyperthermia and thermally responsive nanodrugs such as nCCM for augmented cancer therapy.

Algae-facilitated chemical phosphorus removal during high-density Chlorella emersonii cultivation in a membrane bioreactor.

An algae-based membrane bioreactor (A-MBR) was evaluated for high-density algae cultivation and phosphorus (P) removal. The A-MBR was seeded with Chlorella emersonii and operated at a hydraulic retention time of 1day with minimal biomass wastage for about 150days. The algae concentration increased from initially 385mg/L (or 315mg biomass COD/L) to a final of 4840mg/L (or 1664mg COD/L), yielding an average solids (algae biomass+minerals) production rate of 32.5gm(-3)d(-1) or 6.2gm(-2)d(-1). The A-MBR was able to remove 66±9% of the total P from the water while the algal biomass had an average of 7.5±0.2% extracellular P and 0.4% of intracellular P. The results suggest that algae-induced phosphate precipitation by algae is key to P removal and high-density algae cultivation produces P-rich algal biomass with excellent settling properties.

In vitro neuroprotective potential of four medicinal plants against rotenone-induced toxicity in SH-SY5Y neuroblastoma cells.

BACKGROUND: Lannea schweinfurthii, Zanthoxylum capense, Scadoxus puniceus and Crinum bulbispermum are used traditionally to treat neurological disorders. The aim of this study was to evaluate the cytoprotective potential of the four plants, after induction of toxicity using rotenone, in SH-SY5Y neuroblastoma cells. METHODS: Cytotoxicity of the plant extracts and rotenone was assessed using the sulforhodamine B (SRB) assay. Fluorometry was used to measure intracellular redox state (reactive oxygen species (ROS) and intracellular glutathione content), mitochondrial membrane potential (MMP) and caspase-3 activity, as a marker of apoptotic cell death. RESULTS: Of the tested plants, the methanol extract of Z. capense was the least cytotoxic; LC50 121.3 ± 6.97 µg/ml, while S. puniceus methanol extract was the most cytotoxic; LC50 20.75 ± 1.47 µg/ml. Rotenone reduced intracellular ROS levels after 24 h exposure. Pre-treating cells with S. puniceus and C. bulbispermum extracts reversed the effects of rotenone on intracellular ROS levels. Rotenone exposure also decreased intracellular glutathione levels, which was counteracted by pre-treatment with any one of the extracts. MMP was reduced by rotenone, which was neutralized by pre-treatment with C. bulbispermum ethyl acetate extract. All extracts inhibited rotenone-induced activation of caspase-3. CONCLUSION: The studied plants demonstrated anti-apoptotic activity and restored intracellular glutathione content following rotenone treatment, suggesting that they may possess neuroprotective properties.

Involvement of intracellular glutathione in regulating isoline-induced cytotoxicity in human normal liver L-02 cells.

Pyrrolizidine alkaloid isoline is isolated from the traditional Chinese medicine Ligularia duciformis. Our previous reports have already demonstrated isoline-induced liver injury in mice. The present study is designed to observe the involvement of intracellular reduced glutathione (GSH) in isoline-induced cytotoxicity in human normal liver L-02 cells. The results showed that isoline decreased the cellular GSH and the ratio of GSH and oxidized glutathione in a time- and concentration-dependent manner in L-02 cells. l-Buthionine-S-R-sulfoximine (BSO) is reported to inhibit cellular GSH biosynthesis, and further results showed that isoline decreased the cell viability in L-02 cells after pretreated with 25 µM BSO for 24 h. Furthermore, adducts of isoline and GSH were identified in L-02 cells using liquid chromatography/electrospray ionization tandem mass spectrometry (ion trap) for the first time. In conclusion, our study provides the strongest evidence to support the important roles of GSH in regulating isoline-induced cytotoxicity in human normal liver L-02 cells.

Exposure-dose-response of Anadara trapezia to metal contaminated estuarine sediments: 3. Selenium spiked sediments.

Selenium enters near shore marine environments from the activities of coal-fired power stations. Although selenium is an essential element, at elevated concentrations it can cause genotoxic damage. The relationship between selenium exposure dose and response was investigated in Anadara trapezia exposed to selenium spiked sediment (5 µg/g and 20 µg/g dry mass) for 56 days. A. trapezia reached an equilibrium selenium tissue concentration (2 µg/g and 10 µg/g respectively) by day 42. Gills had significantly more selenium than the hepatopancreas and haemolymph. Between 12 and 21% of accumulated selenium in the gill and hepatopancreas was detoxified and in the metal rich granule. Most of the biologically active selenium in both tissues was in the mitochondrial fraction. Glutathione peroxidase activity and mean total glutathione concentrations for selenium exposed organisms were not significantly different to controls. The ratio of reduced to oxidised glutathione and the total antioxidant capacity were significantly reduced in selenium exposed organisms compared to control organisms. Increased selenium exposure resulted in significant increases in lipid peroxidation, lysosomal destabilisation and an increased frequency of micronuclei. A significant exposure-dose-response relationship for A. trapezia exposed to selenium enriched sediments indicates that elevated sediment selenium concentrations can increased biologically active selenium burdens and cause impairment of cellular processes and cell integrity.

Treatment of Phaeodactylum tricornutum cells with papain facilitates lipid extraction.

Triacylglycerols (TAGs) from microalgae have the potential to be used for biodiesel, but several technical and economic hurdles have to be overcome. A major challenge is efficient extraction of intracellular TAGs from algae. Here we investigate the use of enzymes to deconstruct algal cell walls/membranes. We describe a rapid and simple assay that can assess the efficacy of different enzyme treatments on TAG-containing algae. By this means crude papain and bromelain were found to be effective in releasing TAGs from the diatom Phaeodactylum tricornutum, most likely because of their cysteine protease activity. Pre-treating algal biomass with crude papain enabled complete extraction of TAGs using heptane/isopropyl alcohol. Heptane as a single solvent was also effective, although complete recovery of TAG was not obtained. Economic implications of these findings are discussed, with the aim to reduce the complexity of, and energy needed in, TAG extraction.

Effects of palmitic acid on lipid metabolism homeostasis and apoptosis in goose primary hepatocytes.

Studies have shown that not only does palmitic acid promote triglyceride (TG) accumulation, but it also affects cell viability in in vitro steatosis models. However, to what degree these effects are mediated by steatosis in goose primary hepatocytes is unknown. In this study, the effects of palmitic acid on the lipid metabolism homeostasis pathway and on apoptosis were determined. The authors measured the mRNA levels of genes involved in TG synthesis, lipid deposition, fatty acid oxidation and the assembly and secretion of VLDL-TG in goose primary hepatocytes. The results indicated that palmitic acid can significantly reduce the activity of goose hepatocytes, and that palmitic acid had a significant effect on TG accumulation; however, with increasing palmitic acid concentrations, the extracellular TG and extracellular VLDL concentration gradually decreased. With increasing palmitic acid concentrations, the gene expression levels of DGAT1, DGAT2, PPARα, CPT-1, FoxO1 and MTTP (which regulate hepatic TG synthesis, fatty acid oxidation and the assembly and secretion of VLDL-TGs) first increased and then decreased; the change in PLIN gene expression was palmitic acid dose-dependent, similar to the regulatory mode of intracellular TG accumulation. In conclusion, this study clearly shows that palmitic acid can promote TG accumulation and induce apoptosis in goose primary hepatocytes, and this effect may be related to the lipid metabolism pathway.

Dammarane-type saponins from the flower buds of Panax ginseng and their intracellular radical scavenging capacity.

Korean ginseng (Panax ginseng C.A. Meyer) has been extensively used as a functional food for thousands of years. This study with the aim to evaluate the potential of P. ginseng flower components as a functional food with medicinal properties resulted in the identification of three new dammarane-type saponins, named floralginsenosides Ka-Kc (1-3), along with seventeen known ones (4-20). Their structures were elucidated on the basis of chemical and spectroscopic methods, and their antioxidant activities were evaluated by the intracellular ROS radical scavenging DCF-DA assay. Among them, floralginsenoside Ka (1) displayed potent scavenging activity with the inhibition value of 64% at 10 microM; and ginsenoside Rb(1) (13), floralginsenoside Kc (3), floralginsenoside Kb (2), vinaginsenoside R(9) (11), majoroside F(1) (12), ginsenoside I (17), and ginsenoside II (18) showed moderate scavenging capacity with the inhibition rate of 28, 33, 35, 35, 35, 38, and 38% at 10 microM, respectively. These results warrant further studies concerning the potential of saponin extracts of P. ginseng flowers for functional foods.

Selenium assimilation and loss by an insect predator and its relationship to Se subcellular partitioning in two prey types.

Subcellular selenium (Se) distributions in the oligochaete Tubifex tubifex and in the insect Chironomus riparius did not vary with Se exposure duration, which was consistent with the observations that the duration of prey Se exposure had little influence on either Se assimilation or loss by a predatory insect (the alderfly Sialis velata). However, these two prey types differed in how Se was distributed in their cells. Overall, the predator assimilated a mean of 66% of the Se present in its prey, which was similar to the mean percentage of Se in prey cells (62%) that was theoretically available for uptake (that is, Se in the protein and organelle fractions). Likewise, data for cadmium, nickel and thallium suggest that predictions of trace element transfer between prey and predator are facilitated by considering the subcellular partitioning of these contaminants in prey cells.

In vivo vitamin C supplementation increases phosphoinositol transfer protein expression in peripheral blood mononuclear cells from healthy individuals.

Ascorbate can act as both a reducing and oxidising agent in vitro depending on its environment. It can modulate the intracellular redox environment of cells and therefore is predicted to modulate thiol-dependent cell signalling and gene expression pathways. Using proteomic analysis of vitamin C-treated T cells in vitro, we have previously reported changes in expression of five functional protein groups associated with signalling, carbohydrate metabolism, apoptosis, transcription and immune function. The increased expression of the signalling molecule phosphatidylinositol transfer protein (PITP) was also confirmed using Western blotting. Herein, we have compared protein changes elicited by ascorbate in vitro, with the effect of ascorbate on plasma potassium levels, on peripheral blood mononuclear cell (PBMC) apoptosis and PITP expression, in patients supplemented with vitamin C (0-2 g/d) for up to 10 weeks to investigate whether in vitro model systems are predictive of in vivo effects. PITP varied in expression widely between subjects at all time-points analysed but was increased by supplementation with 2 g ascorbate/d after 5 and 10 weeks. No effects on plasma potassium levels were observed in supplemented subjects despite a reduction of K+ channel proteins in ascorbate-treated T cells in vitro. Similarly, no effect of vitamin C supplementation on PBMC apoptosis was observed, whilst ascorbate decreased expression of caspase 3 recruitment domain protein in vitro. These data provide one of the first demonstrations that proteomics may be valuable in developing predictive markers of nutrient effects in vivo and may identify novel pathways for studying mechanisms of action in vivo.

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.