Use In Toxicity Studies Research Articles

A layered cancer-on-a-chip system for anticancer drug screening and disease modeling.

Recent advances in the development of microfluidic systems for the culture of complex and three-dimensional cell, tissue, and organ models allow their use in toxicity studies and mimicking many diseases. These types of in vitro models are important because of the huge advantages over standard two-dimensional cell cultures: better mimicking of in vivo conditions and more reliable response to the tested drugs. This report presents a new approach to modeling skin cancer (melanoma-on-a-chip) and breast cancer (breast cancer-on-a-chip) using the microfluidic systems. We designed a microfluidic device to co-culture cancer cells with non-malignant cells, which are the main component of the cancer microenvironment. In the construction of the microsystem, we used a scaffold in the form of a porous membrane made of poly(ethylene terephthalate), which enables the regular and reproducible arrangement of cells in the culture and maintains intercellular communication. To demonstrate the functionality of the microsystem, we used it to analyze the effectiveness of photodynamic therapy in the treatment of melanoma and chemotherapy in the treatment of breast cancer. The developed microsystem can be successfully used to model cancer diseases, especially with a layered arrangement of cells in the cancerous tissue, such as melanoma, ductal breast cancer, or breast cancer metastases to the skin.

Immunocytochemistry Analysis of HepG2 Cell 3D Culture Encapsulated as Spheroids in Alginate Beads.

3D Cell culture is an alternative to animal use in many drug development and toxicity studies. The 3D cell culture can mimic and reproduce the original tissue microenvironment, morphology, and mechanical and physiological characteristics, to provide a more realistic and reliable response as compared to two-dimensional cultures. 3D cell culture encapsulated in alginate beads is a very simple and relatively inexpensive tool that is easy to handle and to maintain. The alginate beads function as a scaffold that imprisons cells and allows 3D cell growth, to generate spheroids that can have greater genic expression and cell-cell communication as a nano or microtissue. The HepG2 cell line is a human hepatocellular carcinoma cell derivative. HepG2 cells preserve several of the characteristics of hepatocytes and are therefore often used in toxicity studies. Here, we describe HepG2 cell encapsulation in alginate beads and analyze the resulting spheroids formed within the alginate beads by immunocytochemistry, by staining a certain structure with a specific antibody coupled with a fluorophore. This method preserves the beads and enables cell analysis by confocal microscopy.

Making better use of toxicity studies for human health by extrapolating across endpoints_suppl

Making better use of toxicity studies for human health by extrapolating across endpoints_suppl

Making better use of toxicity studies for human health by extrapolating across endpoints.

To develop and evaluate scientifically robust and innovative approaches for the safety assessment of chemicals across multiple regulatory sectors, the EU Reference Laboratory for alternatives to animal testing (EURL ECVAM) has started a project to explore how to better use the available information, including that from existing animal studies. The aim is to minimize reliance on in vivo testing to avoid redundancy and to facilitate the integration of novel non-animal methods in the regulatory setting with the ultimate goal of designing sustainable testing strategies. In this thought-starter paper, we present a number of examples to illustrate and trigger further discussions within the scientific and regulatory communities on ways to extrapolate useful information for predicting toxicity from one toxicity endpoint to another or across endpoints based on mechanistic information.

Synthesis of ZnO nanoparticles by flame spray pyrolysis and characterisation protocol

There is uncertainty concerning the potential toxicity of zinc oxide (ZnO) nanoparticles, which may be attributed in part to a lack of understanding with regard to the physiochemical properties of the nanoparticles used in toxicological investigations. This paper reports the synthesis of a ZnO nanopowder by flame spray pyrolysis and demonstrates that the typically employed characterisation techniques such as specific surface area measurement and X-ray diffraction provide insufficient information on the sample, especially if it is intended for use in toxicity studies. Instead, a more elaborate characterisation protocol is proposed that includes particle morphology as well as detailed compositional analysis of the nanoparticle surface. Detailed transmission electron microscopy analysis illustrated the polydispersity within the sample: particles were elongated in the c-crystallographic direction, with average Ferret length ~23 nm and Ferret width ~14 nm. Dynamic light scattering (0.1 w/v% in deionised water, pH 7.4) revealed the particles were agglomerated with a modal secondary particle size of ~1.5 μm. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy indicated the presence of carbonate and hydroxide impurities on the surface of the ZnO nanoparticles and an increase of such impurities was observed as the sample was aged, which might influence the nanoparticle dissolution and/or cellular uptake behaviour. These data will be utilised, in order to facilitate the interpretation and understanding of results from toxicological investigations using in vitro cell lines.

Marked toxicity of Albizia bernieri extracts on embryo–larval development in the medaka fish (Oryzias latipes)

Previous phytochemical studies have shown that the plants of the Albizia genus (Fabaceae) contain bioactive saponins, lignans, spermine alkaloids, flavonoids, glycosides phenols and pyridoxine derivatives. Their extracts sometimes display medical properties, but can have also toxic effects. The purpose of our study was to determine the in vivo toxicity of Albizia bernieri seeds in the experimental model of the medaka fish embryo, which is recommended for use in toxicity studies. Our results show clearly that incubating the embryos or larvae of the medaka fish in a medium containing A. bernieri extracts caused a dose-dependent reduction in embryo or larvae survival. Embryos exposed to an extract of A. bernieri displayed cerebral lesions, such as cell lysis and the emergence of lysosomes in the glial tissue. We conclude that when comparing with data obtained with different plant extracts tested on medaka development in our laboratory, A. bernieri displays an unusually high toxicity. Focussing on the cerebral target as well as the fish behaviour could bring more specific informations.

Characterization of silver nanoparticles using flow-field flow fractionation interfaced to inductively coupled plasma mass spectrometry

The ability to detect and identify the physiochemical form of contaminants in the environment is important for degradation, fate and transport, and toxicity studies. This is particularly true of nanomaterials that exist as discrete particles rather than dissolved or sorbed contaminant molecules in the environment. Nanoparticles will tend to agglomerate or dissolve, based on solution chemistry, which will drastically affect their environmental properties. The current study investigates the use of field flow fractionation (FFF) interfaced to inductively coupled plasma-mass spectrometry (ICP-MS) as a sensitive and selective method for detection and characterization of silver nanoparticles. Transmission electron microscopy (TEM) is used to verify the morphology and primary particle size and size distribution of precisely engineered silver nanoparticles. Subsequently, the hydrodynamic size measurements by FFF are compared to dynamic light scattering (DLS) to verify the accuracy of the size determination. Additionally, the sensitivity of the ICP-MS detector is demonstrated by fractionation of μg/L concentrations of mixed silver nanoparticle standards. The technique has been applied to nanoparticle suspensions prior to use in toxicity studies, and post-exposure biological tissue analysis. Silver nanoparticles extracted from tissues of the sediment-dwelling, freshwater oligochaete Lumbriculus variegatus increased in size from approximately 31–46 nm, indicating a significant change in the nanoparticle characteristics during exposure.

Rainbow trout primary epidermal cell proliferation as an indicator of aquatic toxicity: an in vitro/in vivo exposure comparison

Little or no work has been carried out on primary cell cultures in terms of cellular proliferation and toxicity studies. Cell proliferation represents one of the most relevant cellular functions. Anti-PCNA antibodies have aroused considerable interest recently as potential immunocytochemical markers of proliferation for use in toxicity studies. In this study, PCNA methodology, which was developed primarily for mammalian tissues, was adapted to rainbow trout ( Oncorhynchus mykiss (R.)) primary cultured epidermal cells exposed in vivo i.e. whole animal exposures and in vitro for the study of the ecotoxicological potential of the aromatic amine, 2,4-dichloroaniline (2,4-DCA), a member of a little studied and widespread class of aquatic pollutants. There are many approaches to assess the proliferative activity of cells. Immunocytochemical methods offer a high sensitivity and specificity. The immunohistochemical avidin–biotin complex (ABC) method was used for the detection and quantification of PCNA, one of the best-known endogenous proliferation markers, applying the mammalian monoclonal antibody PC-10 to formalin-fixed primary cultures of rainbow trout skin. Here we describe our experience with the immunocytochemical detection and quantification of this proliferation marker. Results indicate that the antibody cross reacts with the corresponding rainbow trout epitope and that the alterations in PCNA labelling in the in vivo and in vitro exposed cultures followed similar patterns. This paper presents data on the validation of rainbow trout primary epidermal culture as an in vitro ecotoxicity model with epidermal proliferation as an endpoint. It can be concluded that cellular proliferation could be used as an indicator of the aquatic toxicity potential of xenobiotics. Correlations between cellular proliferation responses in primary cultures derived from in vivo exposed rainbow trout and primary cultures exposed in vitro were assessed. A dose-response was evidenced in both approaches, however the in vivo exposures appeared to be approximately two orders of magnitude more sensitive than the in vitro exposures. Responses in vitro occurred between 200 and 1000 μM while in vivo responses were between 2 and 10 μM. The good qualitative correspondence between the in vitro and in vivo results indicates that studies using trout epidermal cells allow the identification of xenobiotic effects in fish skin. However, further work is required before quantitative predictions i.e. effective concentrations in vivo, can be made from in vitro studies. This study suggests that the in vitro exposed rainbow trout primary cultured cell model with proliferation as an endpoint can be used as an alternative testing procedure to the whole animal assay.

High-volume, artifact-free fine particle collection for use in toxicity studies

High-volume, artifact-free fine particle collection for use in toxicity studies

Generation and Characterization of Respirable Beryllium Oxide Aerosols for Toxicity Studies

Methods are described to produce respirable particles of 7Be-labeled beryllium oxide at temperature treatments of up to 1000°C for use in toxicity studies. Solutions of beryllium oxalate and suspensions of beryllium hydroxide were nebulized to produce polydisperse particles with activity median aerodynamic diameters in the range of 0.5–0.7 μm and geometric standard deviations between 2.7 and 3.0. Conversion to beryllium oxide was incomplete when the nebulized particles were passed through a tube furnace at temperatures up to 1000° C. Complete conversion was achieved by calcining samples of the collected particles in a muffle furnace for 16 hours at 500 or 1000°C. Particles produced by nebulizing and heat-treating the beryllium chloride or oxalate solutions were hollow shells, but particles nebulized from beryllium hydroxide suspensions were aggregates morphologically similar to industrially produced beryllium oxide particles.

Preparation and characterization of water-soluble fractions of crude and refined oils for use in toxicity studies.

The increase in oil contamination of marine and estuarine environments has led to concern about the effects of oil pollution on marine organisms (CONNELL and MILLER 1981). The most immediate toxic and subtoxic fractions of oils are those soluble in water (HUFFORD 1971), toxicity of oils to organisms usually being related to the content of non-volatile persistent aromatic hydrocarbons (ZIKTO and CARSON 1970r NEFF and ANDERSON 1975). Accumulated evidence indicates that mixing parameters and other factors may play a critical role in determining the quantity of oil and hence the aromatic hydrocarbons transferred to the water column (BOYLAND and TRIPP 1971; GORDON et al. 1973; WINTERS et al. 1976). This paper reports the results of an investigation of factors affecting the preparation of water soluble fractions of oils for use in toxicity studies. The use of synchronous fluorescence spectroscopy for the characterization of fractions was also examined.

Histopathological and Biochemical Effects of Feeding Excess Dietary Methionine to Broiler Chicks

Three experiments were conducted to determine the histological and biochemical effects of toxic levels of dietary DL-methionine on broiler chicks fed an isolated soy-protein/cornstarch-purified diet containing 20% protein, 0.59% methionine, and 3,304 kilocalories of metabolizable energy per kilogram. An appropriate level of supplementary DL-methionine to use in toxicity studies was found to be 1.5%. It significantly depressed (P less than 0.01) gain in body weight, hematocrit, and hemoglobin concentration, increased (P less than 0.05) the level of iron in liver and spleen, caused pancreatic damage, and induced neurological changes. Unlike the retarded growth and increased iron levels in spleen and liver, the fall in hematocrit and hemoglobin values was independent of the reduction in feed intake caused by excess dietary methionine.

An improved experimental medium for freshwater toxicity studies using Daphnia magna

Some of the difficulties involved in formulating experimental media for use in toxicity studies are pointed out, with particular reference to heavy metals and their possible chelation by agents in the media. A medium suitable for Daphnia culture with the green alga, Chlorella sp., was chosen from the literature to investigate physiological responses by Daphnia to sublethal cadmium poisoning. Like most synthetic media this included the labile Fe-EDTA complex as a source of soluble iron which is shown to be essential for growth of both Daphnia and Chlorella. Attention is drawn to the almost univeral complexing ability of EDTA with metal ions, and to the mechanisms whereby EDTA may be released from the Fe-EDTA complex under experimental conditions. Synthesis of a ferri-gluconate complex as an alternative source of soluble iron is described; this complex being theoretically more iron-specific than Fe-EDTA, whilst providing equally well for Chlorella growth and Daphnia growth and fecundity. Restriction of pH variation in the experimental medium, within its chemically stable range of 6.0–9.0, is shown to be difficult over 10 days: most buffers with a suitable p K α value ( ca. 7.5) being toxic or having some complexing ability. TES buffer (p K α 7.5) and HEPES buffer (p K α 7.55) were used at various concentrations, and 0.001 M HEPES is shown to restrict pH variation to within 0.5 pH units over 10 days without adversely affecting Chlorella growth or Daphnia growth and fecundity. Chemical estimation of the degree of cadmium-complexing by each iron source and each buffer under investigation was not possible since the presence of chelating agents imposes restrictions on the methods available, particularly at low cadmium concentrations. Instead, a bioassay of the reduction in 48h acute toxicity of cadmium to Daphnia in media containing each potential chelating agent in turn is used. These data indicate that 0.001 M HEPES buffer does not complex cadmium at 0.1 ppm, and that ferri-gluconate is less likely to do so than ferri-EDTA.

An improved experimental medium for freshwater toxicity studies using Daphnia magna

Some of the difficulties involved in formulating experimental media for use in toxicity studies are pointed out, with particular reference to heavy metals and their possible chelation by agents in the media. A medium suitable for Daphnia culture with the green alga, Chlorella sp. was chosen from the literature to investigate physiological responses by Daphnia to sublethal cadmium poisoning. Like most synthetic media this included the labile Fe-EDTA complex as a source of soluble iron which is shown to be essential for growth of both Daphnia and Chlorella. Attention is drawn to the almost universal complexing ability of EDTA with metal ions, and to the mechanisms whereby EDTA may be released from the Fe-EDTA complex under experimental conditions. Synthesis of a ferri-gluconate complex as an alternative source of soluble iron is described; this complex being theoretically more iron-specific than Fe-EDTA, whilst providing equally well for Chlorella growth and Daphnia growth and fecundity. Restriction of pH variation in the experimental medium, within its chemically stable range of 6.0–9.0, is shown to be difficult over 10 days: most buffers with a suitable p K a value ( ca. 7.5) being toxic or having some complexing ability. TES buffer (p K a 7.5) and HEPES buffer (p K a 7.55) were used at various concentrations, and 0.001 M HEPES is shown to restrict pH variation to within 0.5 pH units over 10 days without adversely affecting Chlorella growth or Daphnia growth and fecundity. Chemical estimation of the degree of cadmium-complexing by each iron source and each buffer under investigation was not possible since the presence of chelating agents imposes restrictions on the methods available, particularly at low cadmium concentrations. Instead, a bioassay of the reduction in 48 h acute toxicity of cadmium to Daphnia in media containing each potential chelating agent in turn is used. These data indicate that 0.001 M HEPES buffer does not complex cadmium at 0.1 ppm, and that ferri-gluconate is less likely to do so than ferri-EDTA.

Some natural factors limiting the abundance of the alfalfa butterfly

does not appear. First page follows. Introduction The early history of the alfalfa butterfly as a pest is well covered in the works of (Wildermuth (1911), (1914), (1920).4 In California and other parts of the West the caterpillar of this butterfly, Colias eurytheme Boisduval,5 is the most serious pest attacking alfalfa. Here, in cases of severe outbreaks, entire fields are often defoliated. During the course of the alfalfa weevil investigation in the region adjacent to the San Francisco Bay and in the northwest portion of the San Joaquin Valley, it was observed that the amount of damage done by the alfalfa butterfly varied not only from year to year but from field to field. One of the reasons for this behavior came to light during September, 1938. In an alfalfa field near Tracy a large number of larvae were collected for use in toxicity studies. The alfalfa was about one third grown and supported a very large population of small larvae. At first it appeared that the developing population was sufficient to inflict serious damage. However, most of the larvae collected proved to be parasitized by a hymenopterous parasite, Apanteles flaviconehae Riley. (Wildermuth (1914)) probably referred to this parasite when he recorded A. fiavicombe as a relatively unimportant parasite of the alfalfa butterfly at Salt Lake City, Utah.