Surface functionalities of gold nanoparticles impact embryonic gene expression responses.

Incorporation of gold nanoparticles (AuNPs) into consumer products is increasing; however, there is a gap in available toxicological data to determine the safety of AuNPs. In this study, we utilised the embryonic zebrafish to investigate how surface functionalisation and charge influence molecular responses. Precisely engineered AuNPs with 1.5 nm cores were synthesised and functionalized with three ligands: 2-mercaptoethanesulfonic acid (MES), N,N,N-trimethylammoniumethanethiol (TMAT), or 2-(2-(2-mercaptoethoxy)ethoxy)ethanol. Developmental assessments revealed differential biological responses when embryos were exposed to the functionalised AuNPs at the same concentration. Using inductively coupled plasma-mass spectrometry, AuNP uptake was confirmed in exposed embryos. Following exposure to MES- and TMAT-AuNPs from 6 to 24 or 6 to 48 h post fertilisation, pathways involved in inflammation and immune response were perturbed. Additionally, transport mechanisms were misregulated after exposure to TMAT and MES-AuNPs, demonstrating that surface functionalisation influences many molecular pathways.

Role of vicinal protein thiols in radiation and cytotoxic responses.

Glutathione (GSH) and more recently protein thiols (P-SH) have been found to play a major role in cellular radiation response. However, the effects of protein vicinal thiols, which are important for the functions of several major enzymes, on cellular responses to radiation have not been clearly delineated. Here we investigated the effects of depleting GSH and protein vicinal thiols (HS-P-SH) and P-SH on cell toxicity and radiation response. We used hydroxyethyldisulfide (HEDS, beta-mercaptoethanol-disulfide) alone and in combination with phenylarsine oxide (PAO) to alter P-SH, HS-P-SH and GSH. HEDS, a direct substrate for thioredoxin reductase and an indirect substrate for glutaredoxin (thioltransferase), did not alter protein vicinal thiols in cells. However, PAO, which specifically forms a covalent adduct with vicinal thiols, blocked bioreduction of HEDS; there was a concomitant and yet unexplained decrease in K1 cell GSH in the presence of HEDS and PAO. G6PD+ (K1) and G6PD- (E89) cells treated with L-buthionine sulfoximine (L-BSO) for 72 h to deplete GSH followed by PAO showed an increased cytotoxic response. However, the surviving E89 cells showed a 10,000-fold greater radiation lethality than the K1 cells. The effects of rapid depletion of GSH by a combination of L-BSO and dimethyfumarate (DMF), a glutathione-S-transferase substrate, were also investigated. Under these conditions, PAO radiosensitized the E89 cells more than 1000-fold over the K1 cells. The potential mechanisms for the altered response may be related to the inhibition of thioredoxin reductase and glutaredoxin. Both are key enzymes involved in DNA synthesis, protein homeostasis and cell survival. With GSH removed, vicinal thiols appear to play a critical role in determining cell survival and radiosensitivity. Decreasing P-SH and removing GSH and vicinal thiols is extremely toxic to K1 and E89 cells. We conclude that radiation sensitivity and cell survival are dependent on vicinal thiol and GSH. In the former and latter cases, the protein thiols are also important.

Electromagnetic re-warming of cryopreserved tissues: effect of choice of cryoprotectant and sample shape on uniformity of heating.

A method that has been proposed for the cryopreservation of tissues and organs is to add a cryoprotective agent (CPA) in sufficient concentration to allow vitrification, and to use rapid electromagnetic heating to prevent the formation of ice crystals during the re-warming. We have compared the physical and biological properties of four CPAs, measuring the speed and uniformity of heating in a 36 mm sphere placed in a 434 MHz applicator, and the toxicity to ECV304 endothelial cells. Ethanediol and dimethyl sulfoxide were found to be suitable for rapid, uniform heating but toxic to the endothelial cells at vitrifying concentrations. Butane-2,3-diol was less toxic, but the heating patterns were unacceptably non-uniform. Propane-1,2-diol was not significantly more toxic than butane-2,3-diol, and did allow uniform heating. It is therefore the best choice of CPA for the vitrification of tissues. We have shown that the uniformity of heating correlates with the dielectric properties of the perfusate. Furthermore, we have shown that uniform heating is feasible in non-spherical samples provided they are approximately ellipsoidal.

[Toxicity of mercaptoethanol to mutant strains of the yeast Pichia methanolica growing on different carbon sources]. / Toksichnost' merkaptoétanola dlia mutantnykh linii drozhzhei Pichia methanolica, rastushchikh na razlichnykh istochnikakh ugleroda.

Addition of beta-mercaptoethanol at a concentration of 2-3 mM to media containing methanol, glucose, or yeast extract caused a 50% inhibition of the growth of wild-type yeast Pichia methanolica; mercaptoethanol at a concentration of 0.7 to 25 mM inhibited the growth of the mutant strain ecr1. The mutation mth1 of P. methanolica repressed its ability to consume methanol and was accompanied by the loss of alcohol oxidase (EC 1.1.3.13) activity. beta-Mercaptoethanol restored the ability of mth1 mutant cells to grow on methanol and stimulated their growth under derepression conditions. The growth effect of beta-mercaptoethanol during derepression was accompanied by partial restoration of alcohol oxidase activity.

Complement activation and inflammation triggered by model biomaterial surfaces.

Biomaterial-mediated complement activation repeatedly has been invoked as a trigger of phagocyte reactions and inflammation. However, a direct correlation between complement activation and inflammatory responses to biomaterial surfaces has yet to be established. Using an animal implantation model and gold surfaces bearing various thiol-linked functionalities, we investigated the potency of different surface groups in prompting complement activation in vitro and surface-mediated accumulation of inflammatory cells in vivo. Among the surfaces tested, mercaptoglycerol- and mercaptoethanol-bearing surfaces engendered the strongest inflammatory responses, as reflected by the accumulation of large numbers of adherent neutrophils and monocytes/macrophages. In contrast, L-cysteine-coated surfaces caused only minor inflammatory responses, and both glutathione-modified and untreated gold implants attracted minimal numbers of inflammatory cells. The accumulation of inflammatory cells on mercaptoglycerol surfaces appears to arise from surface-mediated complement activation because complement-depleted animals failed to exhibit inflammatory responses to mercaptoglycerol-modified implants. Furthermore, there is a close relationship between surface-mediated complement activation (as measured by in vitro iC3b/C5b-9 generation and C3 deposition) and in vivo inflammatory responses. At least in this animal model and with these model surfaces, our results indicate that surface-mediated complement activation can be responsible for the subsequent accumulation of inflammatory cells on implant surfaces.

A trial to compare the effects of pH, buffer concentration, and NaCl, on one fluorescent and two bioluminescent bacterial tests for acute toxicity.

Of all the bioassays to determine acute toxicity described in the literature, those that employ bacteria as indicator organisms are usually the most rapid and the most economic, although alone they cannot predict the possible toxic effect of any type of substance. When bioassays are employed to test the toxicity of known substances and of compounds in samples from waste discharges they have to work in very different conditions from those for which they are designed. The effects of three factors, pH, buffer concentration, and NaCl, on the performance of a fluorogenic bioassay based on the beta-glucuronidase activity of Escherichia coli were investigated. The results of this test were compared with those of two known biluminescent bacteria tests. The fluorogenic bioassay has a more restricted optimum pH range, while the influence of buffer concentration was similar for the three tests. E. coli glucuronidase activity was affected at a concentration as low as 128 mg/l of NaCl. Changes in the pH or buffer concentrations or chloride ions, greatly influenced the respectives toxicities of four substances, acridine orange, TEMED, 2-mercaptoethanol, and mercuric chloride.

Analysis of the six additional chemicals for in vitro assays of the European Economic Communities' EEC aneuploidy programme using Saccharomyces cerevisiae D61.M and the in vitro porcine brain tubulin assembly assay.

We tested six additional chemicals (acetaldehyde, benomyl, diethylstilboestrol, diethylstilboestrol dipropionate, griseofulvin, and mercaptoethanol) for in vitro systems of the coordinated programme to study aneuploidy induction sponsored by the Commission of the European Communities in two in vitro test systems. Using Saccharomyces cerevisiae D61.M (mitotic chromosomal malsegregation assay), benomyl showed a dose-dependent increase in the frequency of chromosomal malsegregation with a lowest effective dose tested (LEDT) of 30 micrograms/ml (0.1 mM). Diethylstilboestrol (DES) showed solvent-dependent effects. DES dissolved in ethanol induced an increase in chromosomal malsegregation as well as in the frequency of total resistant colonies (mutations and recombinations) with a LEDT around 13 micrograms/ml (0.048 mM). Using dimethylsulfoxide as the solvent, no increases were observed with DES up to 333 micrograms/ml (1.24 mM). Acetaldehyde induced an increase in chromosomal malsegregation with the cold treatment protocol (LEDT: 1.25 microliters/ml (21 mM) and 0.75 microliters/ml (13 mM), respectively) but no increase with the overnight protocol (highest dose tested (HDT): 1.75 microliters/ml; 30 mM). Concerning the frequency of total cycloheximide-resistant colonies (mutations and recombinations) increases were obtained with both protocols. The other three compounds were negative when tested up to toxic doses (survival below 10%), up to the maximum solubility in the solvent used or up to heavy precipitation in the incubation mix. The HDT were 333 micrograms/ml (0.88 mM) for diethylstilboestrol dipropionate, 1,600 micrograms/ml (4.5 mM) for griseofulvin and 0.5 microliters/ml (7 mM) for mercaptoethanol. Concerning effects on porcine brain tubulin assembly in vitro, diethylstilboestrol and griseofulvin inhibited the assembly process. The IC30% (30% inhibition concentration) values were 12.5 microM and 100 microM for DES and griseofulvin, respectively. Mercaptoethanol showed no effects up to 50 mM.

Mercaptoethanol and dithiothreitol decrease the difference of electrochemical proton potentials across the yeast plasma and vacuolar membranes and activate their H(+)-ATPases.

Mercaptoethanol and dithiothreitol (DTT) inhibited the acidification of external medium by Saccharomyces carlsbergensis cells and protoplasts during glucose oxidation. The inhibition was also observed when cells were incubated with mercaptoethanol or when mercaptoethanol and DTT were used to prepare protoplasts. Experiments with S. carlsbergensis plasma membrane vesicles and vacuoles showed these thiol reagents to inhibit ATP-dependent generation of delta pH and Em across plasma membrane vesicles and vacuoles but to activate their H(+)-ATPases. Mercaptoethanol and DTT are suggested to de-energize plasmalemma as well as tonoplast by increasing their H(+)-permeability and to disturb the cell ion homeostasis.

Comparative study of intracellular glutathione content in rat lymphocyte cultures treated with 2-mercaptoethanol and interleukin-2.

The level of intracellular glutathione (GSH) in mitogen-stimulated mouse lymphocytes is increased in the presence of 2-mercaptoethanol (2-ME), an enhancer of lymphocyte activation and proliferation. Since proliferation of lymphocytes in response to mitogens involves direct activation by a mitogen followed by continued proliferation in response to interleukin-2 (IL-2), we have investigated the effect of 2-ME and exogenous IL-2 on the GSH content and cell proliferation of rat lymphocytes stimulated with phytohemagglutinin (PHA). PHA stimulation increased both GSH content and the magnitude of the proliferative response, as measured by thymidine incorporation into cellular DNA. However, incubation of stimulated lymphocytes with 2-ME or IL-2 for 72 hr produced a significant further elevation of GSH levels and thymidine incorporation. 2-ME also increased the GSH content in unstimulated cultures, but it had little effect on thymidine incorporation. IL-2 increased GSH content and decreased thymidine incorporation in unstimulated lymphocytes. Exposure of cells to DL-buthionine-(S,R)-sulfoximine (BSO), an inhibitor of GSH biosynthesis, significantly depleted GSH and lowered the proliferative response, suggesting a crucial role of de novo GSH synthesis for lymphocyte activation. The data suggest that both 2-ME and IL-2 promote lymphocyte proliferation, although the mechanisms by which intracellular GSH levels are increased by the agents are apparently different.

Specificity of acute effects of Cd on renal function.

Renal reabsorption of a typical low-molecular weight protein (cytochrome c) by rats was compared in controls and in rats acutely poisoned with CdCl2 (20 mumol/kg i.p., together with 300 mumol mercaptoethanol), HgCl2 (3 mg/kg i.m.), or NiCl2 (10 mg/kg i.p.). The effects of these metals on reabsorption of filtered cytochrome c were expressed in terms of changes induced in total protein excretion and in urinary concentrating ability. On this basis, only Cd exerted an acute specific effect on transport of cytochrome c, similar to its production of low molecular weight proteinuria following chronic exposure. At the time of the experiments the animals contained only low Cd levels in the renal cortex (7 micrograms/g). We conclude that the acutely injected rat can serve at least to some extent as a valid model for the study of Cd nephrotoxicity and that cytochrome c is a useful probe for the study of Cd-induced low molecular weight proteinuria.