Effects of individual terphenyls and polychlorinated terphenyls on rat hepatic microsomal cytochrome P-450-dependent monooxygenases: structure-activity relationships

The effects of o-, m- and p-terphenyl, 2,4-dichloro-, 2,4,6-trichloro-, 2,3,5,6-tetrachloro-, 2,3,4,6-tetrachloro-, 2,4,4'",6- tetrachloro- and 2,3,4,5-tetrachloro-p-terphenyl, 2,3,4,5-tetrachloro-m- and o-terphenyl as inducers of hepatic drug-metabolizing enzymes were determined in immature male Wistar rats. o-Terphenyl, 2,4-dichloro-, 2,4,6-trichloro-p-terphenyl and 2,3,4,5-tetrachloro-o-terphenyl induced 4,4-dimethylamino antipyrine N-demethylase at total dose levels of 300 mol/kg and the 2,3,4,5-tetrachloro-p-terphenyl induced ethoxyresorufin O-deethylase (EROD). In contrast, none of the other terphenyls or polychlorinated terphenyls (PCTs) induced these enzyme activities. Previous studies have demonstrated that 2,3,4,5-tetrachloro-p-terphenyl did not exhibit a high affinity for the 2,3,7,8-tetrachlorodibenzo-p-trachlorodibenzo-p-dioxin (TCDD) receptor protein (EC₅₀= 6.6×10^–6M). In contrast, this study showed that 2,3,4,5-tetrachloro-p-terphenyl was more active than either 2,3,4,5-tetrachloro-o- or m-terphenyl as an inducer of EROD. Moreover, the competitive receptor binding EC₅₀ values for the latter two isomers were > 10^–5 M and this result was also consistent with their lack of EROD induction activity. Previous studies showed that analysis of the data for a series of 4-substituted-2,3,4,5-tetrachlorobiphenyls indicated that the p-terphenyl structural moiety (i.e. 4-substituent = phenyl) did not interact with high affinity with the receptor protein binding site. Since the 2,3,4,5-tetrachloro o- and m-terphenyls are also poor ligands for the receptor protein, this data and results from other studies indicate that PCT congeners (and commercial mixtures) are therefore unlikely to elicit significant 2,3,7,8-TCDD-like biologic or toxic effects in target species.     

Immunoglobulin G from patients with heparin-induced thrombocytopenia binds to a complex of heparin and platelet factor 4

Heparin-induced thrombocytopenia (HIT) is an important complication of heparin therapy. Although there is general agreement that platelet activation in vitro by the HIT IgG is mediated by the platelet Fc receptor, the interaction among the antibody, heparin, and platelet membrane components is uncertain and debated. In this report, we describe studies designed to address these interactions. We found, as others have noted, that a variety of other sulfated polysaccharides could substitute for heparin in the reaction. Using polysaccharides selected for both size and charge, we found that reactivity depended on two independent factors: a certain minimum degree of sulfation per saccharide unit and a certain minimum size. Hence, highly sulfated but small (< 1,000 daltons) polysaccharides were not reactive nor were large but poorly sulfated polysaccharides. The ability of HIT IgG to recognize heparin by itself was tested by Ouchterlony gel diffusion, ammonium sulfate and polyethylene glycol precipitation, and equilibrium dialysis. No technique demonstrated reactivity. However, when platelet releasate was added to heparin and HIT IgG, a 50-fold increase in binding of radio-labeled heparin to HIT IgG was observed. The releasate was then depleted of proteins capable of binding to heparin by immunoaffinity chromatography. Only platelet factor 4-immunodepleted releasate lost its reactivity with HIT IgG and heparin. Finally, to determine whether the reaction occurred on the surface of platelets or in the fluid phase, washed platelets were incubated with HIT IgG or heparin and after a wash step, heparin or HIT IgG was added, respectively. Reactivity was only noted when platelets were preincubated with heparin. Consistent with these observations was the demonstration of the presence of PF4 on platelets using flow cytometry. These studies indicate that heparin and other large, highly sulfated polysaccharides bind to PF4 to form a reactive antigen on the platelet surface. HIT IgG then binds to this complex with activation of platelets through the platelet Fc receptors.     

Amino-acid substitution in the disordered loop of blood group B-glycosyltransferase enzyme causes weak B phenotype

BACKGROUND: Few studies have investigated the reaction kinetics and interactions with nucleotide donor and acceptor substrates of mutant human ABO glycosyltransferases. Previous work identified a B(w) allele featuring a 556G>A polymorphism giving rise to a weak B phenotype. This polymorphism is predicted to cause a M186V amino-acid mutation within a highly conserved series of 16 amino acids present both in both blood group A- and blood group B-synthesizing enzymes. These residues are known as the disordered loop because their location cannot be determined in the crystal structure of the enzyme. Another patient has been identified with a 556G>A B(w) allele and the kinetics of the resulting mutant glycosyltransferase were studied. STUDY DESIGN AND METHODS: Serologic testing with murine and human reagents, amplification of the coding regions of exons 6 and 7, and DNA sequencing were performed with standard protocols. Enzyme kinetic studies utilized a model of human GTB M186V expressed in Escherichia coli with radiolabeled UDP-galactose and UDP-N-acetylgalactosamine as donor substrates and synthetic H-disaccharide as acceptor following standard protocols. RESULTS: The patient's red blood cells demonstrated a weak, but not mixed-field, B phenotype. Kinetic studies on the mutant enzyme revealed diminished activity (k(cat) = 0.15 per sec with UDP-galactose compared to 5.1 per sec for wild-type GTB) and elevated K(m) values for all substrates. CONCLUSION: This enzyme with a mutation in the disordered loop produces weak B antigen expression because of greatly decreased enzyme activity and reduced affinity for B-donor and acceptor substances.