Assessment of the eye irritation potential of chemicals: A comparison study between two test methods based on human 3D hemi-cornea models.

We have recently developed two hemi-cornea models (Bartok et al., Toxicol in Vitro 29, 72, 2015; Zorn-Kruppa et al. PLoS One 9, e114181, 2014), which allow the correct prediction of eye irritation potential of chemicals according to the United Nations globally harmonized system of classification and labeling of chemicals (UN GHS). Both models comprise a multilayered epithelium and a stroma with embedded keratocytes in a collagenous matrix. These two models were compared, using a set of fourteen test chemicals. Their effects after 10 and 60 minutes (min) exposure were assessed from the quantification of cell viability using the MTT reduction assay. The first approach separately quantifies the damage inflicted to the epithelium and the stroma. The second approach quantifies the depth of injury by recording cell death as a function of depth. The classification obtained by the two models was compared to the Draize rabbit eye test and an ex vivo model using rabbit cornea (Jester et al. Toxicol in Vitro. 24, 597-604, 2010). With a 60 min exposure, both of our models are able to clearly differentiate UN GHS Category 1 and UN GHS Category 2 test chemicals.

Epidermal tight junctions in health and disease.

The skin, the largest organ of the body, is an essential barrier that under homeostatic conditions efficiently protects and/or minimizes damage from both environmental (e.g. microorganisms, physical trauma, ultraviolet radiation) and endogenous (e.g., cancers, inflammation) factors. This formidable barrier function resides mainly in the epidermis, a dynamic, highly-stratified epithelium. The epidermis has 2 major barrier structures: stratum corneum, the outmost layer and tight junctions, intercellular junctions that seal adjacent keratinocytes in the stratum granulosum, found below the stratum corneum. In recent years there have been significant advances in our understanding of tight junction function, composition and regulation. Herein we review what is known about tight junctions in healthy skin and keratinocyte culture systems and highlight the dynamic crosstalk observed between tight junctions and the cutaneous immune system. Finally we discuss the preliminary observations suggesting that tight junction function or protein expression may be relevant for the pathogenesis of a number of common cutaneous inflammatory and neoplastic conditions.

Development of an in vitro ocular test system for the prediction of all three GHS categories.

In the present study we considered a new approach that allows the individual quantification of damages induced in the epithelium and stroma of an in vitro hemi-cornea model after chemical treatment. We aimed at a stand-alone test system for classification according to the classification of the globally harmonized system of classification and labelling of chemicals (GHS). We have modified a previously developed 3D hemi-cornea model by the insertion of a collagen membrane between epithelium and stroma. This membrane allows the separation and independent assessment of these compartments after topical exposure to potential eye irritants. The cell viability quantified by MTT assay was used as the toxicological endpoint. The prediction model based on the results obtained from 30 test chemicals uses a single exposure period and the combination of cut-off values in tissue viability from both epithelium and stroma. The in vitro-in vivo concordance of the test system is 77%. All of the GHS category 1, 80% of the GHS category 2 and 50% of the GHS not categorized chemicals are predicted correctly. In conclusion, the test system predicts and discriminates GHS category 1 and GHS category 2 chemicals, but is over-predictive for GHS not categorized materials.

A human hemi-cornea model for eye irritation testing: quality control of production, reliability and predictive capacity.

We have developed a 3-dimensional human hemi-cornea which comprises an immortalized epithelial cell line and keratocytes embedded in a collagen stroma. In the present study, we have used MTT reduction of the whole tissue to clarify whether the production of this complex 3-D-model is transferable into other laboratories and whether these tissues can be constructed reproducibly. Our results demonstrate the reproducible production of the hemi-cornea model according to standard operation procedures using 15 independent batches of reconstructed hemi-cornea models in two independent laboratories each. Furthermore, the hemi-cornea tissues have been treated with 20 chemicals of different eye-irritating potential under blind conditions to assess the performance and limitations of our test system comparing three different prediction models. The most suitable prediction model revealed an overall in vitro-in vivo concordance of 80% and 70% in the participating laboratories, respectively, and an inter-laboratory concordance of 80%. Sensitivity of the test was 77% and specificity was between 57% and 86% to discriminate classified from non-classified chemicals. We conclude that additional physiologically relevant endpoints in both epithelium and stroma have to be developed for the reliable prediction of all GHS classes of eye irritation in one stand alone test system.

Evaluation of the cytotoxicity of selected systemic and intravitreally dosed drugs in the cultures of human retinal pigment epithelial cell line and of pig primary retinal pigment epithelial cells.

The cytotoxicity of the selected systemic and intravitreally dosed drugs tamoxifen, toremifene, chloroquine, 5-fluorouracil, gentamicin and ganciclovir was studied in retinal pigment epithelium (RPE) in vitro. The cytotoxicity was assayed in the human RPE cell line D407 and the pig RPE cell culture using the WST-1 test, which is an assay of cell proliferation and viability. The effects of experimental conditions on the WST-1 test (cell density, serum content in the culture medium, the exposure time) were evaluated. The EC50 values in tamoxifen-treated D407 cells ranged between 6.7 and 8.9 micromol/l, and in pig RPE cells between 10.1 and 12.2 micromol/l, depending on the cell density used. The corresponding values for toremifene were 7.4 to 11.1 micromol/l in D407 cells and 10.0 to 11.6 micromol/l in pig RPE cells. In chloroquine-treated cells, the EC50 values were 110.0 micromol/l for D407 cells and 58.4 micromol/l for pig RPE cells. Gentamicin and ganciclovir did not show any toxicity in micromolar concentrations. The exposure time was a significant factor, especially when the drug did not induce cell death, but was antiproliferative (5-fluorouracil). Serum protected the cells from the toxic effects of the drugs. Both cell cultures were most sensitive to tamoxifen and toremifene, and next to chloroquine. The drug toxicities obtained in the present study were quite similar in both cell types; that is, the pig RPE cells and the human D 407 cell line, despite the differences in, for example, the growth rate and melanin contents of the cell types. Owing to the homeostatic functions important for the whole neuroretina, RPE is an interesting in vitro model for the evaluation of retinal toxicity, but, in addition to the WST-1 test, more specific tests and markers based on the homeostatic functions of the RPE are needed.

Carotenoid incorporation into natural membranes from artificial carriers: liposomes and beta-cyclodextrins.

Liposomes and beta-cyclodextrin (beta-CD) have been used as carriers for the incorporation of three dietary carotenoids (beta-carotene (BC), lutein (LUT) and canthaxanthin (CTX)) into plasma, mitochondrial, microsomal and nuclear membrane fractions from pig liver cells or the retinal epithelial cell line D407. The uptake dynamics of the carotenoids from the carriers to the organelle membranes and their incorporation yield (IY) was followed by incubations at pH 7.4 for up to 3 h. The mean IYs saturated between 0.1 and 0.9 after 10-30 min of incubation, depending on membrane characteristics (cholesterol to phospholipid ratio) and carotenoid specificity. Mitochondrial membranes (more fluid) favour the incorporation of BC (non-polar), while plasma membranes (more rigid) facilitate the incorporation of lutein, the most polar carotenoid. A high susceptibility of BC to degradation in the microsomal suspension was observed by parallel incubations with/without 2,6-di-t-buthyl-p-cresol (BHT) as antioxidant additive. The beta-CD carrier showed to be more effective for the incorporation of lutein while BC was incorporated equally into natural membranes either from liposomes or from cyclodextrins. The presence of cytosol in the incubation mixture had no significant effects on the carotenoid incorporations.

Gene dosage affects the expression of the duplicated NHP6 genes of Saccharomyces cerevisiae.

Nhp6Ap and Nhp6Bp, which are 87% identical in sequence, are moderately abundant, chromosome-associated proteins from Saccharomyces cerevisiae. In wild type cells Nhp6Ap is present at three times the level of Nhp6Bp. The effects of altering NHP6A or NHP6B gene number on the expression of its partner has been examined using Northern blots and reporter genes. Deletion of NHP6A led to a three-fold increase in NHP6B synthesis while an extra copy of NHP6A reduced NHP6B expression two-fold. Changes in the NHP6B gene copy number caused more moderate changes in NHP6A synthesis. The regulation of one NHP6 gene by the other uses a mechanism that detects the level of Nhp6 protein (or RNA) rather than gene number, since overexpression of Nhp6B protein from a single gene led to a dramatic decrease in NHP6A synthesis. Deletion analysis showed that the regulatory element involved in gene dosage compensation maps to a 190 bp segment in the NHP6B promoter. The simplest model, that each Nhp6 protein can act as a transcriptional repressor at the other NHP6 gene, is not true since purified Nhp6A protein does not bind specifically to the NHP6B promoter region. Instead, Nhp6p appears to interact with or through another protein in regulating transcription from the NHP6 genes.

Nhp6, an HMG1 protein, functions in SNR6 transcription by RNA polymerase III in S. cerevisiae.

Nhp6A and Nhp6B are HMG1-like proteins required for the growth of S. cerevisiae at elevated temperatures. We show that the conditional lethality of an nhp6 strain results from defective transcription of SNR6 (U6 snRNA) by RNA polymerase III. Overexpression of U6 snRNA or Brf1, a limiting component of TFIIIB, and an activating mutation (PCF1-1) in TFIIIC were each found to suppress the nhp6 growth defect. Additionally, U6 snRNA levels, which are reduced over 10-fold in nhp6 cells at 37 degrees C, were restored by Brf1 overexpression and by PCF1-1. Nhp6A protein specifically enhanced TFIIIC-dependent, but not TATA box-dependent, SNR6 transcription in vitro by facilitating TFIIIC binding to the SNR6 promoter. Thus, Nhp6 has a direct role in transcription complex assembly at SNR6.

Mutations in the yeast Nhp6 protein can differentially affect its in vivo functions.

Nhp6A and Nhp6B from S. cerevisiae are required for viability at 38 degrees C because they are involved in transcription of SNR6 by RNA polymerase III. Nhp6A also represses transcription of NHP6B by RNA polymerase II. Nhp6 belongs to the HMG1 family, defined by an 80 amino acid DNA binding domain, which includes six highly conserved residues. These amino acids were mutated in Nhp6A and their affects on Nhp6 function were assessed in vivo. Surprisingly, most of the changes allowed Nhp6A to function normally in supporting growth at 38 degrees C. However, six mutants had differential effects on in vivo function. Finally, two of the mutant proteins that did not restore Nhp6A function in vivo were shown to bind and bend DNA in vitro as well as wild type. Together, these results suggest that Nhp6 interacts with another protein(s) to carry out some of its biological functions and that this interaction might differ at promoters transcribed by RNA polymerase II versus RNA polymerase III.

Membrane-permeant, bioactivatable analogues of cGMP as inducers of cell death in IPC-81 leukemia cells.

We report an improved single-step synthesis to generate the membrane-permeant acetoxymethyl esters (AM-esters) of cGMP and three cGMP-analogues. These bioactivatable compounds were found to induce cell death in rat IPC-81 cells, a model system for acute myelocytic leukemia, in micromolar doses, while the corresponding non-modified cGMP-analogues were inactive.