Cytotoxicity assay to assess eye irritation - A comparison with other methods and possible strategies for use.

Many in vitro methods can be used to classify eye irritation or damage caused by exposure to a substance. In this study, a recently described method called the Cytotoxicity Assay to Assess Eye Irritation (CEI) was compared with other selected in vitro methods adopted in the OECD guidelines. Furthermore, the influence of combining more than one in vitro method was investigated. Basic performance indices were considered and a risk assessment based on the number of correct and incorrect results (overestimated or underestimated) was performed. The CEI results were similar to those of other in vitro tests, however, the CEI can also directly classify substances irritating to the eyes. When the CEI preceded an eye irritation test based reconstructed human corneal-like epithelium, there were fewer underestimates compared with other method combinations. This combination can better protect human health and provides results comparable to those obtained in animal tests.

Developing a screening test for toxicity studies of prenatal development with the use of Hydra attenuata and embryos of zebrafish.

11 active substances used in pesticides were selected. Substances were divided into three groups depending the effect on embryos or fetuses of laboratory animals: 1 - damaging effect on embryos or fetuses (embryotoxic, fetotoxic or teratogenic), 2 - damaging effect on embryos or fetuses, but only at dose toxic for mother (maternal toxicity), 3 - no damaging effect. Changes for hydra in acute toxicity tests and recovery tests were assessed on an change scale from 0 to 10. The index of the effect on development (TI) for hydras was calculated for every compound. Changes in zebrafish embryos were assessed using a descriptive method. Pearson correlation coefficient showed the correlation between the concentration and the toxic effect in the zebrafish embryos for the substances of the first group. The study showed that substances having a strong damaging effect on fetuses cause changes that are apparent and easy to evaluate both in hydras and zebrafish embryos. A scoring system was introduced to evaluate the changes of hydras and zebrafish embryos. The point system of evaluation of changes allows quick classification of a substance as potentially embryotoxic, fetotoxic or teratogenic. It allows developing a cheap and fast method alternative to prenatal developmental toxicity studies, a screening method that enables substances of great teratogenic potential to be excluded from studies on laboratory animals.

A proposal for a new in vitro method for direct classification of eye irritants by cytotoxicity test - Preliminary study.

None of the in vitro method are suitable for directly classifying of a substance as an eye irritant (category 2). They can classify substance as category 1 (serious eye damage) or as "no category" (not requiring classification). The aim of this study was to develop a new method for direct classification of a substance as category 2. Cytotoxicity Assay to Assess Eye Irritation (CEI) was performed on fibroblast - HDFn cell line with 36 substances. 5 concentrations of all substances and neat substances were applied directly to the cells. After 30 min, medium was added and cells were incubated at 37 °C. The next day, the cytotoxicity assay was performed (MTT assay in the first run and NRU assay in the second run). Based on viability and IC50 value (concentration with 50 % viability) a substance could be classified in category 2, category 1, and as "no category". The results obtained were referred to ECHA database. This new method had high sensitivity (53.8-88.9 %), specificity (73.9-100.0 %) and accuracy (69.4-88.9 %) in the classification to all categories. It effectively classifies not only substances in category 2 but also in category 1 and substances that do not require classification.

Heterogeneous Mixture of Amniotic Cells is Likely a Better Source of Stem Cells than Adipose Tissue.

Stem cells are increasingly being used in the course of burn treatment. As several different types of stem cells are available for the purposes, it is important to chose the most efficient and the most practicable stem cell type. The aim of this study was to compare the potential of heterogeneous amnion cell mixture with the presently used standard therapy, the adipose tissue-derived stem cells. The placenta was collected during a Cesarean section procedure. Adipose tissue tissue-derived cells were isolated using the Cytori's Celution® System. Cells were tested for fulfillment of the minimum criteria for stem cells. The efficiency of cell cultures was tested by an analysis of population doubling, cell proliferation, cell cycle and cell migration. Amniotic cells presented a higher ability for differentiation to chondrocytes and osteocytes than adipose-derived regenerative cells but a lower ability for differentiation toward adipocytes. Additionally, in vitro experiments have demonstrated a higher applicability of amniotic cells than adipose tissue-derived stem cells. Amniotic cells show several advantages: easy access to placenta, low costs and a lack of ethical dilemmas related to stem cell harvesting. The main disadvantage is, however, their availability, as isogenic treatment would only be possible for women around children-bearing age, unless personalized banks for amniotic cells would be established.

Notch signaling pathway and gene expression profiles during early in vitro differentiation of liver-derived mesenchymal stromal cells to osteoblasts.

Notch signaling is a key signaling pathway for cell proliferation and differentiation. Therefore, we formulated a working hypothesis that Notch signaling can be used to detect early osteoblastic differentiation of mesenchymal stromal cells. Changes in expression and distribution of Notch 1, 2, 3, and Delta1 in the cytoplasm and nuclei of rat liver-derived mesenchymal stromal cells differentiating into osteoblasts were investigated, together with the displacement of intracellular domains (ICDs) of the receptors. In addition, an oligonucleotide microarray was used to determine the expression of genes known to be linked to selected signaling pathways. Statistically significant changes in the number of cells expressing Notch1, Notch2, and Delta1, but not Notch3, and their activated forms were detected within 24 h of culture under osteogenic conditions. Although the number of cells expressing Notch3 remained unchanged, the number of cells with the activated receptor was significantly elevated. The number of cells positive for Notch3 was higher than that for the other Notch receptors even after 48 h of differentiation; however, a smaller fraction of cells contained activated Notch3. Culture mineralization was detected on day 4 of differentiation, and all analyzed receptors were present in the cells at that time, but only Delta1 was activated in twice as many cells than that before differentiation. Thus, the three analyzed receptors and ligand can serve as markers of very early stages of osteogenesis in stromal cells. These early changes in activation of the Notch signaling pathway were correlated with the transcription of several genes linked to osteogenesis, such as Bmps, Mmps, and Egfr, and with the regulation of cell cycle and apoptosis.

Application of EPR spectroscopy to the examination of pro-oxidant activity of coffee.

Free radicals present in coffee may be responsible for exerting toxic effects on an organism. The objectives of this work were to compare free radicals properties and concentrations in different commercially available coffees, in solid and liquid states, and to determine the effect of roasting on the formation of free radicals in coffee beans of various origins. The free radicals content of 15 commercially available coffees (solid and liquid) was compared and the impact of processing examined using electron paramagnetic resonance (EPR) spectroscopy at X-band (9.3 GHz). First derivative EPR spectra were measured at microwave power in the range of 0.7-70 mW. The following parameters were calculated for EPR spectra: amplitude (A), integral intensity (I), and line-width (ΔBpp); g-Factor was obtained from resonance condition. Our study showed that free radicals exist in green coffee beans (10(16) spin/g), roasted coffee beans (10(18) spin/g), and in commercially available coffee (10(17)-10(18) spin/g). Free radical concentrations were higher in solid ground coffee than in instant or lyophilised coffee. Continuous microwave saturation indicated homogeneous broadening of EPR lines from solid and liquid commercial coffee samples as well as green and roasted coffee beans. Slow spin-lattice relaxation processes were found to be present in all coffee samples tested, solid and liquid commercial coffees as well as green and roasted coffee beans. Higher free radicals concentrations were obtained for both the green and roasted at 240 °C coffee beans from Peru compared with those originating from Ethiopia, Brazil, India, or Colombia. Moreover, more free radicals occurred in Arabica coffee beans roasted at 240 °C than Robusta. EPR spectroscopy is a useful method of examining free radicals in different types of coffee.