Differential binding affinities of PCBs, HO-PCBs, and aroclors with recombinant human, rainbow trout (Onchorhynkiss mykiss), and green anole (Anolis carolinensis) estrogen receptors, using a semi-high throughput competitive binding assay.
A comparative study was undertaken to assess the ability of 44 polychlorinated biphenyls (PCBs), 9 hydroxylated PCBs (HO-PCBs), and 8 aroclors at concentrations ranging from 1 nM to 10 microM to compete with [3H]17beta-estradiol (E2) for binding to bacterially expressed fusion proteins using a semi-high throughput competitive-binding assay. The fusion proteins consisted of the D, E, and F domains of human (alpha), cloned reptilian (Anolis carolinensis) and recloned rainbow trout (Onchorhynkiss mykiss) estrogen receptors (ER) linked to the glutathione S-transferase (GST) protein. GST-hERalphadef (human), GST-aERdef (reptile) and GST-rtERdef (rainbow trout) fusion proteins exhibited high affinity for E2 with dissociation constants (Kd) of 0.4+/-0.1 nM, 0.7+/-0.2 nM, and 0.6+/-0.1 nM, respectively. Of the 44 PCBs examined, only PCBs 104, 184, and 188 effectively competed with [3H]E2 for binding to the GST-rtERdef protein with IC50 values ranging from 0.4-1.3 microM. In contrast, these same congeners only caused a 30% displacement of [3H]E2 in GST-hERalphadef and GST-aERdef proteins. Several additional congeners were found to bind to the GST-rtERdef fusion protein, although the degree of interaction varied among congeners. Among the HO-PCBs, 2',3',4',5'-tetrachloro-4-biphenylol and 2,6,2',6'-tetrachloro-4-biphenylol bound to all three fusion proteins with IC50 values ranging from 0.1-0.3 microM. Dimethyl sulphoxide (DMSO) concentrations of 20% significantly increased the ability of PCBs 104, 184, and 188 to compete with [3H]E2 for binding to the GST-ERdef fusion proteins, whereas at 20% DMSO, a significant reduction in saturable binding was observed. These results demonstrate that ERs from different species exhibit differential ligand preferences and relative binding affinities for PCBs, which can be dramatically affected by DMSO concentration.
Matthews
J
J
Department of Biochemistry and National Food Safety and Toxicology Center, Michigan State University, East Lansing 48824-1319, USA.
Zacharewski
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001140:Aroclors / Q000378:metabolism*; D001483:Base Sequence; D001667:Binding, Competitive; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000032:analysis / Q000302:isolation & purification; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008116:Lizards; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D011078:Polychlorinated Biphenyls / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011960:Receptors, Estrogen / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*
D001140:Aroclors; D017931:DNA Primers; D018076:DNA, Complementary; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D011078:Polychlorinated Biphenyls; D005982:Glutathione Transferase