The thyroid hormone (TH)-disrupting activity of effluents and environmental water samples in Thailand was surveyed by three in vitro bioassays with different endpoints. These assays test the potency of competitive binding with the active form of TH, 3,3',5-[(125)I]triiodo-l-thyronine (T(3)), to the plasma transport protein transthyretin (TTR) and TH receptor (TR; the TTR assay and TR assay, respectively) and the interference with the cellular T(3)-signaling pathway through TR-mediated luciferase gene activation (the luc assay). The TH-disrupting activity in water samples collected from paper manufacturing plants (PMPs), the canal Khlong U-Taphao, and a sewage-treatment plant (STP) was detected predominantly in the dichloromethane/methanol or methanol fractions of solid-phase extraction, suggesting a similar hydrophobic nature of the causative contaminants. The TR assay was relatively more sensitive than the TTR assay to the competitively potent contaminants. The luc assay indicated that the dichloromethane/methanol fractions of most water samples contained anti-T(3)-like activity. Our assays demonstrated that wastewater treatment effectively removed the TH-disrupting contaminants from wastewater in the PMP and the STP. The potencies for TH disruption at the three sampling points of the STP exhibited positive correlations among the three bioassays, whereas those from the canal and PMP water were not correlated among the three bioassays. Furthermore, the influent contaminants that were competitively potent in the TTR assay partially affected the luc assay. These bioassays are useful monitoring tools that give results relevant for evaluating the health of amphibian populations.
We have investigated the sulfation of thyroid hormones (THs) in the cytosol from Rana catesbeiana tadpole tissues. Sulfation of 3,3',5-triiodothyronine (T(3)) by the liver cytosol, which was dependent on protein amount, incubation time, and temperature, suggested the presence of TH sulfotransferases (SULTs) in the liver. The apparent Michaelis-Menten constant (K(m)) of the liver cytosol was 0.22 microM for T(3), and the apparent maximum velocity (V(max)) of the liver cytosol was 7.65 pmol/min/mg protein for T(3). Iodothyronine sulfating activity in the liver cytosol was increased in tadpoles at premetamorphic (stages IX-X) and metamorphic climax (stage XX) stages, and in adult frogs. The substrate preference of iodothyronine sulfation for the liver cytosol from tadpoles (stage X) was: 3,3',5'-triiodothyronine>T(3)>3,3',5,5'-tetraiodothyroacetic acid>3,3',5-triiodothyroacetic acid, T(4), 3-iodothyronine>3,5-diiodothyronine. Several halogenated phenols were potent inhibitors (IC(50)=0.15-0.21 microM). The substrate preference for T(3) was gradually lost by the onset of metamorphic climax stages. These enzymatic characteristics of iodothyronine sulfation in the liver cytosol from tadpoles resembled those of mammalian phenol SULTs, except that the tadpole cytosol had a higher affinity (one or two orders of magnitude) for T(3) than mammalian SULTs. These results suggested that an enzyme homologous to mammalian phenol SULT (SULT1) may be involved in TH metabolism in tadpoles.
Aldehyde dehydrogenases (ALDHs) convert aldehydes into their corresponding carboxylic acids. ALDH1A1, also known as ALDH class 1 (ALDH1) or retinaldehyde dehydrogenase (RALDH1), prefers retinal to acetaldehyde as a substrate. To investigate the effects of divalent cations on the dehydrogenase activity of Xenopus laevis ALDH1A1, the formation of acetate and retinoic acid from acetaldehyde and retinal, respectively, was investigated in the presence of Ca2+, Mg2+, Mn2+ or Zn2+. All divalent cations tested inhibited the oxidation of acetaldehyde and retinal by ALDH1A1. When acetaldehyde was used as a substrate, the 50% inhibitory concentrations (IC50) were 10, 24, 35 and 220 microM for Zn2+, Mn2+, Mg2+ and Ca2+, respectively. Kinetic studies of ALDH1A1 dehydrogenase activity in the presence or absence of each cation revealed that the inhibition mode by cations was uncompetitive against acetaldehyde, retinal, and NAD+, and that their inhibitory potencies were greater against acetaldehyde than retinal. It was concluded that the divalent cations inhibited X. laevis ALDH1A1 activity in a substrate-dependent manner by affecting a step of the dehydrogenase reaction that occurred after the formation of the ternary complex of the enzyme, substrate, and coenzyme.
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