Screening of some anti-androgenic endocrine disruptors using a recombinant cell-based in vitro bioassay.

The present work describes the development and optimization of a cell-based androgen reporter assay using the Chinese hamster ovarian cell line (CHO K1) in the 96-well format. The recent reports on increasing exposure of humans and wild-life to environmental endocrine disrupting chemicals (ED) prompt the need for high throughput screening systems for such compounds in environmental and biological samples. To this end, CHO cells were cotransfected with plasmids encoding mouse mammary tumour virus-neomycin-luciferase and human androgen receptor (hAR), and a stable cell line was established. After selection with neomycin, a highly active clone was obtained which stably expressed both the hAR and the androgen-responsive luciferase reporter. Stimulation of the cells with androgens for 24 h resulted in about 15-fold stimulation of luciferase activity, with the minimum effective dose of testosterone being 0.1 nmol/l. Potent steroidal and non-steroidal anti-androgens, such as hydroxyflutamide and cyproterone acetate, significantly inhibited the androgen-induced transactivation. Non-androgenic steroids like estradiol, progesterone, dexamethasone and cortisol showed weak activity at high concentrations. RT-PCR and western blot confirmed proper transcription and translation as well as stable expression of the AR gene in the cells. About 60 different chemicals (mostly pesticides or their metabolites, and common industrial chemicals) were screened with the cell line for their ability to stimulate luciferase activity or inhibit that evoked by 0.1 nmol/l R1881, used as a positive androgenic control. About 10 highly potent anti-androgenic chemicals were identified. The most potent anti-androgenic compounds identified in our assay included bisphenol A, alpha-hexachlorocyclohexane, vinclozolin and 4,4-DDE. These compounds had alone either no effect or were weak agonists (with cytotoxic effects at very high concentrations), but none showed any significant agonistic activity. In conclusion, we demonstrate that the bioassay based on this cell line provides a reliable test for detecting androgenic and anti-androgenic compounds. The 96-well plate format makes the assay suitable for high throughput screening.

Identification of a novel cytokine-like transcript differentially expressed in avian gammadelta T cells.

Chicken represents a species with a high frequency of gammadelta T cells in peripheral blood, suggesting an important function. To elucidate the genes specific for the avian gammadelta T cells, the suppression subtractive hybridization (SSH) between gammadelta and alphabeta T cells was used. The SSH library, which was successfully enriched for the TCR gamma and delta (both V and C region) sequences, provided gammadelta T-cell-specific genes, including, for example, the ribosomal proteins, signaling and structural molecules, and molecules related to transcription and translation. Among these genes, a clone named KK34 was shown to match the PFAM profile for IL-5 and to have 19.5% amino acid identity to the human interleukin 5 protein. Clone KK34 had lower sequence homology, from 5.4% to 15.6%, to other short-chain four-helix bundle superfamily members IL-3, IL-4, IL-13 and GM-CSF. The hydrophobic signal peptide sequence and the presence of cysteines needed for the interchain disulfide bonds were found to be conserved between clone KK34 and mammalian IL-5 proteins. Clone KK34 transcript expression was studied by RT-PCR, Northern blotting and in situ hybridization and it was confirmed to be expressed in avian gammadelta T cells. We propose that this clone, KK34, may represent the first nonmammalian IL-5, supporting the findings that gammadelta T cells are important in the development of allergy.

Ultrastructural characterization of developmental and degenerative vitreo-retinal changes in the eyes of transgenic mice with a deletion mutation in type II collagen gene.

PURPOSE: Molecular genetic analyses have clearly associated vitreoretinal degeneration with mutations in the type II collagen gene, but lack of experimental models has prevented systematic analyses of the occurrence of phenotypic changes and of the pathogenetic mechanisms involved. The present study is a detailed morphological and ultrastructural analysis of the vitreoretinal consequences of a small deletion mutation in the type II collagen gene. METHODS: The eyes of Del1 mice carrying six copies of pro alpha1(II) collagen transgene with a small deletion mutation were analyzed during embryonic development, postnatal growth and aging using their nontransgenic littermates as controls. Tissue samples were processed for light and electron microscopy for morphological and ultrastructural analyses. Transcription of pro alpha1(II) collagen gene was localized by in situ hybridization, and type II collagen was detected by immunohistochemistry. RESULTS: In this mouse model most components of the eye are ultrastructurally unaltered. However, the transgenes caused a dose-dependent dominant negative effect seen as a reduced number of type II collagen fibrils in the vitreous. In concert with this, dose-dependent accumulation of amorphous material was observed in the dilated rough endoplasmic reticulum of cells responsible for the production of type II collagen molecules. In mice homozygous for the transgene locus, the vitreoretinal degenerative lesions appeared already during late embryonic development. In mice heterozygous for the locus, such changes were milder and appeared only during postnatal growth and progressed gradually upon aging. CONCLUSIONS: The observed ultrastructural changes suggest that defective structure and function of collagen fibrils in Del1 mice result from a partial block in the post-translational processing and secretion of the mutated procollagen chains, and partly from secretion of mutated procollagen molecules which interfere with normal fibrillogenesis.