Cytotoxicity of Halogenated Tyrosyl Compounds, an Emerging Class of Disinfection Byproducts.

Halogenated amino acids and peptides are an emerging class of disinfection byproducts (DBPs), having been detected in drinking water and in washed food products. However, the toxicological significance of these emerging DBPs remains unclear. In this study, the cytotoxicity of eight halogenated tyrosyl compounds was investigated in Chinese hamster ovary (CHO) cells using real-time cell analysis (RTCA). Dihalogenated tyrosyl compounds are more cytotoxic than their monohalogenated analogues. The cytotoxicity of the dihalogenated compounds is associated with their ability to induce intracellular reactive oxygen species (ROS), suggesting that oxidative stress is an important toxicity pathway of these compounds. Pearson correlation analysis of the cytotoxicity (IC50 values) of these compounds with eight physicochemical parameters showed strong associations with their lipophilicity (logP) and reactivity (polarizability, ELUMO). Finally, cytotoxicity testing of the concentrated extracts of a chloraminated mixture of eight dipeptides with bromide or iodide showed the cytotoxicity of these mixtures in the order: iodinated peptides > brominated peptides ≥ chlorinated peptides. These results demonstrate that halogenated peptide DBPs are toxicologically relevant, and further research is needed to understand the implications of long-term exposure for human health.

A microelectric cell sensing technique for in vitro assessment of ocular irritation.

The animal-based Draize test remains the gold standard for assessment of ocular irritation. However, subjective scoring methods, species differences, and animal welfare concerns have spurred development of alternative test methods. In this study, a novel in vitro method for assessing ocular irritancy was developed using a microelectric cell sensing technology, real-time cell analysis (RTCA). The cytotoxicity of sixteen compounds was assessed in two cell lines: ARPE-19 (human retina) and SIRC (rabbit cornea). In vitro inhibitory (IC50 and AUC50) values were determined at 6, 12, 24, 48, 72, and 96 h exposure, with a subset of values confirmed with MTT testing. The values displayed comparable predictivity of in vivo ocular irritation on the basis of a linear regression between the calculated values and each compounds' corresponding Draize-determined modified maximum average score (MMAS), but the ARPE-19 derived values were more strongly correlated than those from SIRC cells. Hence, IC50 values derived from ARPE-19 cells were used to predict the UN GHS/EU CLP classification of each test compound. The method was determined to have sensitivity of 90%, specificity of 50%, and overall concordance of 75%. Thus, RTCA testing may be best incorporated into a top-down tiered testing strategy for identification of ocular irritants in vitro.

Lipid phosphate phosphatases regulate signal transduction through glycerolipids and sphingolipids.

Lipid phosphate esters including lysophosphatidate (LPA), phosphatidate (PA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) are bioactive in mammalian cells and serve as mediators of signal transduction. LPA and S1P are present in biological fluids and activate cells through stimulation of their respective G-protein-coupled receptors, LPA(1-3) and S1P(1-5). LPA stimulates fibroblast division and is important in wound repair. It is also active in maintaining the growth of ovarian cancers. S1P stimulates chemotaxis, proliferation and differentiation of vascular endothelial and smooth muscle cells and is an important participant in the angiogenic response and neovessel maturation. PA and C1P are believed to act primarily inside the cell where they facilitate vesicle transport. The lipid phosphates are substrates for a family of lipid phosphate phosphatases (LPPs) that dramatically alter the signaling balance between the phosphate esters and their dephosphorylated products. In the case of PA, S1P and C1P, the products are diacylglycerol (DAG), sphingosine and ceramide, respectively. These latter lipids are also bioactive and, thus, the LPPs change signals that the cell receives. The LPPs are integral membrane proteins that act both inside and outside the cell. The "ecto-activity" of the LPPs regulates the circulating and locally effective concentrations of LPA and S1P. Conversely, the internal activity controls the relative accumulation of PA or C1P in response to stimulation by various agonists thereby affecting cell signaling downstream of EDG and other receptors. This article will review the various LPPs and discuss how these enzymes could regulate signal transduction by lipid mediators.