Evaluation of tire tread particle toxicity to fish using rainbow trout cell lines.

Tire and road wear particles (TRWP) resulting from tire abrasion while driving raise concerns due to their potential contribution to aquatic toxicity. Our study aimed to assess cryogenically milled tire tread (CMTT) particle toxicity, used as a proxy for TRWP, and associated chemicals to fish using two Rainbow Trout (Oncorhynchus mykiss) cell lines representing the gill (RTgill-W1) and the intestinal (RTgutGC) epithelium. CMTT toxicity was evaluated through several exposure pathways, including direct contact, leaching, and digestion, while also assessing the impact of particle aging. Following OECD TG249, cell viability was assessed after 24 h acute exposure using a multiple-endpoint assay indicative of cell metabolic activity, membrane integrity and lysosome integrity. In vitro EC50 values for the fish cell lines exceeded river TRWP concentrations (2.02 g/L and 4.65 g/L for RTgill-W1 and RTgutGC cell lines, respectively), and were similar to in vivo LC50 values estimated at 6 g/L. Although toxicity was mainly driven by the leaching of tire-associated chemicals, the presence of the particles contributed to the overall toxicity by inducing a continuous leaching, highlighting the importance of considering combined exposure scenarios. Aging and digestion conditions were also found to mediate CMTT toxicity. Thermooxidation resulted in a decreased chemical leaching and toxicity, while in vitro digestion under mimicked gastrointestinal conditions increased leaching and toxicity. Specific chemicals, especially Zn, 2-mercaptobenzothiazole, 1,3-diphenylguanidine, and N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) were identified as contributors to the overall toxicity. Although 6PPD-quinone was detected in CMTT digestate, cytotoxicity assays with RTgill-W1 and RTgutGC cell lines showed no toxicity up to 6 mg/L, supporting the notion of a specific mode of action of this chemical. This study provides insights into the toxicological mechanisms induced by tire particles and their associated chemicals and can help in the evaluation of potential risks to aquatic life associated with TRWP.

Conductive composite fibres from reduced graphene oxide and polypyrrole nanoparticles.

Continuous composite fibres composed of polypyrrole (PPy) nanoparticles and reduced graphene oxide (rGO) at different mass ratios were fabricated using a single step wet-spinning approach. The electrical conductivity of the composite fibres increased significantly with the addition of rGO. The mechanical properties of the composite fibres also improved by the addition of rGO sheets compared to fibres containing only PPy. The ultimate tensile strength of the fibres increased with the proportion of rGO mass present. The elongation at break was greatest for the composite fibre containing equal mass ratios of PPy nanoparticles and rGO sheets. L929 fibroblasts seeded onto fibres showed no reduction in cell viability. To further assess toxicity, cells were exposed to media that had been used to extract any aqueous-soluble leachates from developed fibre. Overall, these composite fibres show promising mechanical and electrical properties while not significantly impeding cell growth, opening up a wide range of potential applications including nerve and muscle regeneration studies.

Effects of clofibric acid alone and in combination with 17ß-estradiol on mRNA abundance in primary hepatocytes isolated from rainbow trout.

Clofibric acid (CA) is the active substance of lipid lowering drugs. It is resistant to degradation, polar in nature, and has been found ubiquitously in the aquatic environment. Though CA is classified as a peroxisomal proliferator in rodents and is considered as a potential endocrine disruptor, little information exists on the effects of CA in aquatic organisms, such as fish. In the present study, we examined the mRNA levels of peroxisome proliferator- and estrogen-sensitive genes on the exposure of primary rainbow trout (Oncorhynchus mykiss) hepatocytes to CA alone and in combination with the natural female sex hormone, 17ß-estradiol (E2). Our results demonstrate that rainbow trout hepatocytes are relatively refractory to the effects of CA on the PPAR signaling pathway and lipid metabolism. Moreover, CA did not show recognizable estrogenic activity, but after the induction of vitellogenesis by E2, CA significantly reduced vitellogenin (VTG) mRNA abundance. Apparently, the indirect repression of VTG transcription, independent of estrogen receptors, occurred. The mechanism is not yet clearly understood but may involve disruption of the stabilization of VTG mRNA known to be induced by E2.

Engineering a multimodal nerve conduit for repair of injured peripheral nerve.

Injury to nerve tissue in the peripheral nervous system (PNS) results in long-term impairment of limb function, dysaesthesia and pain, often with associated psychological effects. Whilst minor injuries can be left to regenerate without intervention and short gaps up to 2 cm can be sutured, larger or more severe injuries commonly require autogenous nerve grafts harvested from elsewhere in the body (usually sensory nerves). Functional recovery is often suboptimal and associated with loss of sensation from the tissue innervated by the harvested nerve. The challenges that persist with nerve repair have resulted in development of nerve guides or conduits from non-neural biological tissues and various polymers to improve the prognosis for the repair of damaged nerves in the PNS. This study describes the design and fabrication of a multimodal controlled pore size nerve regeneration conduit using polylactic acid (PLA) and (PLA):poly(lactic-co-glycolic) acid (PLGA) fibers within a neurotrophin-enriched alginate hydrogel. The nerve repair conduit design consists of two types of PLGA fibers selected specifically for promotion of axonal outgrowth and Schwann cell growth (75:25 for axons; 85:15 for Schwann cells). These aligned fibers are contained within the lumen of a knitted PLA sheath coated with electrospun PLA nanofibers to control pore size. The PLGA guidance fibers within the nerve repair conduit lumen are supported within an alginate hydrogel impregnated with neurotrophic factors (NT-3 or BDNF with LIF, SMDF and MGF-1) to provide neuroprotection, stimulation of axonal growth and Schwann cell migration. The conduit was used to promote repair of transected sciatic nerve in rats over a period of 4 weeks. Over this period, it was observed that over-grooming and self-mutilation (autotomy) of the limb implanted with the conduit was significantly reduced in rats implanted with the full-configuration conduit compared to rats implanted with conduits containing only an alginate hydrogel. This indicates return of some feeling to the limb via the fully-configured conduit. Immunohistochemical analysis of the implanted conduits removed from the rats after the four-week implantation period confirmed the presence of myelinated axons within the conduit and distal to the site of implantation, further supporting that the conduit promoted nerve repair over this period of time. This study describes the design considerations and fabrication of a novel multicomponent, multimodal bio-engineered synthetic conduit for peripheral nerve repair.

Moving targets, long-lived infrastructure, and increasing needs for integration and adaptation in water management: an illustration from Switzerland.

Switzerland provides an example of successful management of water infrastructure and water resources that was accomplished largely without integration across sectors. Limitations in this approach have become apparent; decisions that were formerly based only on technical and economic feasibility must now incorporate broader objectives such as ecological impact. In addition, current and emerging challenges relate to increasingly complex problems that are likely to demand more integrated approaches. If such integration is to be of benefit, it must be possible to redirect resources across sectors, and the synergies derived from integration must outweigh the additional cost of increased complexity.

Applications and potential uses of fish gill cell lines: examples with RTgill-W1.

Gills are unique structures involved in respiration and osmoregulation in piscinids as well as in many aquatic invertebrates. The availability of the trout-derived gill cell line, RTgill-W1, is beginning to make impacts in fish health and toxicology. These cells are available from the American Type Culture Collection as ATCC CRL 2523. The cells have an epithelioid morphology and form tight monolayer sheets that can be used for testing epithelial resistance. The cells can be grown in regular tissue culture surfaces or in transwell membranes in direct contact with water on their apical surfaces. The ability of RTgill-W1 to withstand hypo- and hyper-osmotic conditions and their optimal growth capacity at room temperature, make these cells ideal sentinel models for in vitro aquatic toxicology as well as model systems to study fish gill function and gill diseases. RTgill-W1 support growth of paramyxoviruses and orthomyxoviruses like salmon anemia virus. RTgill-W1 also support growth of Neoparamoeba pemaquidensis, the causative agent of amoebic gill disease. The cells have been used to understand mechanisms of toxicity, ranking the potencies of toxicants, and evaluating the toxicity of environmental samples. These cells are also valuable for high throughput toxicogenomic and toxicoproteomic studies which are easier to achieve with cell lines than with whole organisms. RTgill-W1 cell line could become a valuable complement to whole animal studies and in some cases as gill replacements in aquatic toxicology.

Who is chasing whom? A call for a more integrated approach to reduce the load of micro-pollutants in the environment.

One of the key questions arising from the presence of micro-pollutants in surface-, ground-, and drinking water is whether they pose a risk to human and ecosystem health. In our laboratories we have identified a number of biological effects by several pharmaceuticals and personal care products (PPCPs) on human, animal and/or plant cells at different levels of biological organisation. In part, these effects occur at concentrations even below those reported in drinking water. Even though it is often still difficult to fully deduce the role of some of these effects on the whole organism or population level as well as after chronic exposure, the effects observed illustrate that the input of micro-pollutants into the environment must be avoided or as far as possible reduced. Much effort has already been devoted to improved treatment of sewage and raw drinking water. A comprehensive protection from aquatic micro-pollutants, however, cannot reside in water treatment technology alone. Instead, all components of the life cycle of these chemicals must be put to the table to turn around the current trend of increasing environmental loads. The goal of this report is to illustrate why a more comprehensive way of risk assessment is needed and what this should include.

Mass fluxes and spatial trends of xenobiotics in the waters of the city of Halle, Germany.

The behaviour and the effects of xenobiotics including pharmaceuticals and fragrances in the environment are widely unknown. In order to improve our knowledge, field investigations and modelling approaches for the entire area of the city of Halle/Saale, Germany, were performed. The distribution of the concentration values and mass fluxes are exemplified using indicators such as Bisphenol A, t-Nonylphenol, Carbamacepine, Galaxolide, Tonalide, Gadolinium and isotopes. Concentrations at a magnitude of ng/L to microg/L were found ubiquitously in the ground and surface waters. Using the concentration values, the impact of the city concerning the indicators was not always evident. Only the assessment of the mass fluxes shows significant urban impacts along the city passage. The calculation of the mass fluxes shows increasing values for all investigated xenobiotics during the city passage; only Bisphenol A stagnates. A balance model of water and indicator mass fluxes was built up for the entire city area.

Biophysical and biochemical properties of a binary lipid mixture for DNA transfection.

The phase and miscibility behavior of a triple-chain phosphatidylcholine (TPHPC) and a single-chain surfactant (CTAB) were investigated in aqueous dispersions and in monolayers at the air/water interface. CTAB can be incorporated in the TPHPC monolayer because of its complementary molecule shape and reduces the tilt angle of TPHPC. The type of phases and the phase sequence (L2 - LS) are the same in the pure TPHPC monolayer and in the TPHPC/CTAB (80:20 mol:mol) mixture. No indication of any ordering of adsorbed DNA was observed. In the aqueous dispersions, TPHPC exhibits an inverted hexagonal phase above the chain melting. The addition of 30 mol% CTAB leads to the appearance of a lamellar Lalpha phase. The binding of DNA to the mixture is obvious but this is accompanied by a separation of the two lipids what is supported by monolayer experiments. The system has no long-term stability. The main reason seems to be not only the stronger interaction of DNA with CTAB, but also especially the unexpected weak interaction between CTAB and TPHPC. The transfection efficiency is lower compared with lipofectamine. The main disadvantage of this system is the cytotoxicity of CTAB, which could not be lowered by incorporation of CTAB in the TPHPC bilayer.

Inflammatory activity in river-water samples.

Contamination of the urban aquatic environment with chemical and biological substances could have a long-term impact on human health because these substances threaten the integrity of the urban ecosystem and the availability of high-quality water for recreation and consumption. In light of this, the aim of the present study was to assess the potential immunological effects of water sampled at various sites along the River Saale near the city of Halle (in the state of Sachsen-Anhalt, Germany). For the control, Ficoll-separated peripheral blood mononuclear cells (PBMC) of healthy donors were cultured for 24 h in either filter-sterilized river water or drinking-water samples. Cell vitality was assessed using the MTT bioassay. Cytokines in culture supernatants were measured by ELISA. Endotoxin concentrations in the water samples were assessed by the limulus amoebocyte lysate (LAL) test. River water and drinking water showed comparably weak cytotoxic effects on PBMC. Drinking water did not exert any effect on cytokine secretion. In contrast, all river-water samples triggered secretion of proinflammatory cytokines, as shown for TNF-alpha, IL-1beta, and IL-6. Free endotoxin was detected in all river-water samples. However, the highest inflammatory activity regarding induction of all three cytokines, as well as the highest endotoxin content as determined by LAL, was found in a water sample taken immediately downstream of a wastewater treatment plant. Inhibition studies using the monoclonal anti-CD14 antibody biG14, which is known to suppress binding of lipopolysaccharide (LPS) to CD14 via binding CD14 itself, revealed that free endotoxin was indeed the major inducer of proinflammatory cytokines in the river-water samples. Taken together, the results suggest that the microorganism-derived endotoxin is a widely distributed contaminant in the urban aquatic environment that should be considered in routine monitoring and in assessing ecosystem and human health.

Effect-directed identification of oxygen and sulfur heterocycles as major polycyclic aromatic cytochrome P4501A-inducers in a contaminated sediment.

Heterocyclic polyaromatic compounds, including dinaphthofurans, 2-(2-naphthalenyl)benzothiophene, methylated chrysene, and benz[a]anthracene, were identified and confirmed as major cytochrome P4501A (CYP1A)-inducing compounds in a contaminated sediment close to the industrial site of Bitterfeld (Germany). Identification was achieved by the application of an effect-directed fractionation and analysis approach. This approach comprised the combination of a rainbow trout liver cell line (RTL-W1) bioassay to select for CYP1A-inducing effects by measuring 7-ethoxyresorufin-O-deethylase activity, a multistep fractionation procedure, and various methods of chemical characterization. The identified nonpriority pollutants were found to be significantly more potent than the reference compound, benzo[a]pyrene, and among the most potent polycyclic inducers known. On the basis of the history of industrial activity at the contaminated site, the heterocyclic compounds identified in this study are thought to stem from 2-naphthol production. 2-Naphthol is one of the most high-tonnage products of the aniline dye industry in general, thereby indicating the potential environmental relevance of the identified heterocyclic aromatic compounds. To date, however, no or very little knowledge exists about their occurrence, fate, and biological effects.

Ability of fractionated petroleum refinery effluent to elicit cyto- and photocytotoxic responses and to induce 7-ethoxyresorufin-O-deethylase activity in fish cell lines.

The ability of fractionated petroleum refinery effluent to cause cellular responses in fish cell lines was evaluated. The cellular responses, which included direct and indirect cytotoxicity, photocytotoxicity and induction of 7-ethoxyresorufin-O-deethylase (EROD) activity, may potentially be linked to sublethal effects observed in effluent-exposed fish and fish larvae. In order to be able to quantify cellular responses rapidly, microtitre plates were used along with fluorescent probes. For the quantification of cyto- and photocytotoxicity, the fluorescent probes were alamar Blue and carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM), which monitor metabolic activity and cell membrane integrity, respectively. EROD activity was measured as the rate of conversion by EROD of the substrate 7-ethoxyresorufin to its fluorescent product, resorufin. Effluent from an Ontario refinery was fractionated into aqueous and particulate phase. As well, a solid phase extract (SPE) was used to prepare concentrated effluent for testing in the cell lines. The effluent was able to elicit all of the responses of interest although significant cyto- and photocytotoxicity required effluent equivalent concentrations above 100% effluent and could only be revealed upon exposure of cells to the SPE concentrated effluent. Based on their retention on C18, the cytotoxicants are likely to be non-polar to moderately polar chemicals. The presence of polar compounds affecting cellular metabolism was indicated by the responses of exposed cells to a 90% aqueous phase effluent. In contrast to cyto- and photocytotoxicity, EROD induction occurred at effluent equivalent concentrations well below 100% effluent and was elicited by the SPE and the particulate fraction thereby suggesting that most EROD-inducers were particle-bound. Among other applications, the described techniques could help to determine the source of causative agents of sublethal effects in the refining process.

Polycyclic aromatic hydrocarbons as inducers of cytochrome P4501A enzyme activity in the rainbow trout liver cell line, RTL-W1, and in primary cultures of rainbow trout hepatocytes.

In order to investigate cell-specific differences in the response of in vitro models to environmental toxicants, we compared the capacity of nine polycyclic aromatic hydrocarbons (PAHs) to induce cytochrome P4501A (CYPIA) in primary rainbow trout (Oncorhynchus mykiss) hepatocytes and a rainbow trout liver cell line, RTL-W1. Induction of CYPIA was estimated from the catalytic activity of 7-ethoxyresorufin-O-deethylase (EROD) and compared by median effective concentration (EC50) values, induction spans, and benzo[a]pyrene induction equivalency factors for inducing PAHs. The influence of culture conditions was investigated with respect to the presence or absence of serum and varying exposure times. Both in vitro systems lead to an identical classification of the PAHs in noninducing (anthracene, fluoranthene, phenanthrene, and pyrene) and inducing compounds with a similar ranking of inducing PAHs. Mean EC50 values in RTL-W1 cells were, respectively, 343 and 266 nM for benzo[a]anthracene, 57 and 92 nM for BaP, 134 and 283 nM for benzo[b]fluoranthene, 455 and 270 nM for chrysene, and 98 and 116 nM for 3-methylcholanthrene. Compared to primary hepatocytes, the RTL-W1 cell line was more sensitive in its EROD response to the presence or absence of serum and to the increase in exposure time, which led to higher EC50 values.

Transitory metabolic disruption and cytotoxicity elicited by benzo[a]pyrene in two cell lines from rainbow trout liver.

Two cell lines, RTL-W1 and R1, from rainbow trout liver were used to investigate the effects of benzo[A]pyrene (BaP). BaP induced a catalytic measure of CYP1A, 7-ethoxyresorufin-O-deethylase (EROD) activity, in the rainbow trout liver cell line RTL-W1 but not in R1. Geldanamycin inhibited EROD induction by BaP. Potential BaP metabolites, BaP-7,8-dihydrodiol (BDP) and 6,12-BaP quinone (BQ) also induced EROD activity in RTL-W1. Very low BaP concentrations slightly stimulated cell proliferation in both cell lines. Higher BaP concentrations caused cytotoxicity in RTL-W1 but not in R1. Cytotoxicity was detected in a cell viability assay with 5-carboxyfluorescein diacetate acetoxymethyl ester, and as a decline in cell number. In both cell lines, BaP exposure impaired the reduction of the redox dye, alamar Blue (AB). After BaP removal, AB reduction recovered. Similar results were observed with BQ. As AB monitors metabolic activity, this novel phenomenon was termed transitory metabolic disruption. This decline in AB readings that was caused by BaP was ameliorated in RTL-W1 by alpha-naphthoflavone and geldanamycin, which suggests a role for CYP1A, and in R1 by indomethacin, which suggests involvement of prostaglandin-H-synthase. The significance of the response to BaP that is detected with AB and whether other PAHs cause it will be interesting future questions.

Optical properties of rainbow trout lenses after in vitro exposure to polycyclic aromatic hydrocarbons in the presence or absence of ultraviolet radiation.

The optical properties of rainbow trout lenses were investigated after in vitro exposure to polycyclic aromatic hydrocarbons (PAHs) and ultraviolet (UV) irradiation, both because PAHs frequently contaminate aquatic environments and because UV exposure has generally increased with the decline of the ozone layer. Lenses were exposed to UV irradiation for 12 hr while immersed in culture medium. UV irradiation, with or without the presence of PAHs, was accomplished with one UVA and one UVB photoreactor lamp to yield a photon fluence rate of 9.27 micromol m(-2)s(-1)UVA (UVA:UVB 10.8, radiant exposure of 13.4 Jcm(-1)). Individual PAHs studied were fluorene, fluoranthene and benzo(a)pyrene. In addition, lenses were exposed to a solution of creosote, a wood preservative used in the aquatic environment that contains many PAHs. All PAH exposures, including creosote, were carried out either in the dark or concurrently with UV irradiation. A scanning laser monitor system was used to evaluate the optical properties of lenses for up to 236 hr after the UV/PAH treatments. Mean focal length variability (FLV) increased with time after concurrent exposure to UV irradiation and high concentrations of either fluoranthene (4900 n m), benzo(a)pyrene (265 n m) or creosote (70 microg ml(-1)), with FLV values ranging from, 0.21-0.41, 0.21-0.64 and 0.15-0.22 mm, respectively, 72 hr after termination of the UV/PAH treatment. UV irradiation alone or exposure to PAHs in the dark brought about no changes in the optical properties of lenses. Also, fluorene in the presence or absence of UV had no effect, even at concentrations as high as 128 microm. Lenses were also unchanged by 12 hr exposures in the dark to solutions of either fluorene, fluoranthene, benzo(a)pyrene or creosote that had been previously UV irradiated for 12 hr. This meant that photomodified products of the individual PAHs or creosote were not cataractogenic and emphasized that simultaneous exposure to UV and PAHs or creosote was necessary for the increased FLV. The results point for the first time to an interaction between UV irradiation and PAHs as a potential contributing factor to cataract formation in fish.

[Effectiveness of Formica rufa and autologous blood injection in patients with ankylosing spondylitis: a double-blind randomized study]. / Wirksamkeit von Formica rufa und Eigenblut-Injektionen bei Patienten mit ankylosierender Spondylitis: eine doppelblinde, randomisierte Studie.

While complementary medicine and homeopathy is becoming an increasingly prominent part of the health care practices, there is a lack of controlled studies concerning their effectiveness. In our study, we wanted to answer the question whether a combination of Formica rufa D6 and reinjection of the patient's own blood is superior to injection of placebo. 104 patients with ankylosing spondylitis entered a prospective, randomized, double-blind study. During four weeks they received twice weekly either 1 ml of Formica rufa D6 in combination with 0.5 ml of blood or 1.5 ml NaCl intramuscular. Before and after therapy, mobility, thoracic excursion and doctor's overall assessment were measured in addition to patient's health status, using a German version of the Arthritis Impact Measurement Scales, before and after therapy as well as four, twelve, and twenty-four weeks later. We were not able to detect any statistical difference between treatment and placebo group in any of the parameters measured. Therefore, the therapy under study may not be regarded effective.

Moisture in the granules.

This article offers manufacturers who process a broad range of plastics tips on how to handle polymers that are sensitive to moisture. It covers detection and drying procedures that will help ensure good-quality mouldings are produced.

Transient induction of 7-ethoxyresorufin-O-deethylase (EROD) activity by medium change in the rainbow trout liver cell line, RTL-W1.

Cytochrome P4501A (CYP1A) metabolizes a wide array of lipophilic xenobiotics. In fish liver, CYP1A is constitutively expressed at low levels, but xenobiotics can strongly induce CYP1A expression via a receptor-mediated pathway. While induction of hepatic CYP1A in teleosts by xenobiotics is well investigated, very little is known on the regulation of constitutive CYP1A expression and its induction by factors other than xenobiotics. In the present study we show that in the rainbow trout liver cell line, RTL-W1, CYP1A-catalyzed 7-ethoxyresorufin-O-deethylase (EROD) activity can be induced by a change of the culture medium, in the absence of xenobiotics. The increase in cellular EROD levels is of transient nature. Experiments with cell incubation solutions supplemented with various medium components indicate that photooxidized tryptophan is the agent causing the increase of EROD activity after medium change.