An In Vitro Comparative Study of the Effects of Tetrabromobisphenol A and Tetrabromobisphenol S on Human Erythrocyte Membranes—Changes in ATP Level, Perturbations in Membrane Fluidity, Alterations in Conformational State and Damage to Proteins

Brominated flame retardants (BFRs) are substances used to reduce the flammability of plastics. Among this group, tetrabormobisphenol A (TBBPA) is currently produced and used on the greatest scale, but due to the emerging reports on its potential toxicity, tetrabromobisphenol S (TBBPS)—a compound with a very similar structure—is used as an alternative. Due to the fact that the compounds in question are found in the environment and in biological samples from living organisms, including humans, and due to the insufficient toxicological knowledge about them, it is necessary to assess their impacts on living organisms and verify the validity of TBBPA replacement by TBBPS. The RBC membrane was chosen as the research model. This is a widely accepted research model for assessing the toxicity of xenobiotics, and it is the first barrier to compounds entering circulation. It was found that TBBPA and TBBPS caused increases in the fluidity of the erythrocyte membrane in their hydrophilic layer, and conformational changes to membrane proteins. They also caused thiol group elevation, an increase in lipid peroxidation (TBBPS only) and decreases in the level of ATP in cells. They also caused changes in the size and shape of RBCs. TBBPA caused changes in the erythrocyte membrane at lower concentrations compared to TBBPS at an occupational exposure level.

High-throughput genotyping assays for identification of glycophorin B deletion variants in population studies

Glycophorins are the most abundant sialoglycoproteins on the surface of human erythrocyte membranes. Genetic variation in glycophorin region of human chromosome 4 (containing GYPA, GYPB, and GYPE genes) is of interest because the gene products serve as receptors for pathogens of major public health interest, including Plasmodium sp., Babesia sp., Influenza virus, Vibrio cholerae El Tor Hemolysin, and Escherichia coli. A large structural rearrangement and hybrid glycophorin variant, known as Dantu, which was identified in East African populations, has been linked with a 40% reduction in risk for severe malaria. Apart from Dantu, other large structural variants exist, with the most common being deletion of the whole GYPB gene and its surrounding region, resulting in multiple different deletion forms. In West Africa particularly, these deletions are estimated to account for between 5 and 15% of the variation in different populations, mostly attributed to the forms known as DEL1 and DEL2. Due to the lack of specific variant assays, little is known of the distribution of these variants. Here, we report a modification of a previous GYPB DEL1 assay and the development of a novel GYPB DEL2 assay as high-throughput PCR-RFLP assays, as well as the identification of the crossover/breakpoint for GYPB DEL2. Using 393 samples from three study sites in Ghana as well as samples from HapMap and 1000 G projects for validation, we show that our assays are sensitive and reliable for genotyping GYPB DEL1 and DEL2. To the best of our knowledge, this is the first report of such high-throughput genotyping assays by PCR-RFLP for identifying specific GYPB deletion types in populations. These assays will enable better identification of GYPB deletions for large genetic association studies and functional experiments to understand the role of this gene cluster region in susceptibility to malaria and other diseases.