Characterization of a propylthiouracil-insensitive type I iodothyronine deiodinase

Mammalian type I iodothyronine deiodinase (D1) activates and inactivates thyroid hormone by outer ring deiodination (ORD) and inner ring deiodination (IRD), respectively, and is potently inhibited by propylthiouracil (PTU). Here we describe the cloning and characterization of a complementary DNA encoding a PTU-insensitive D1 from teleost fish (Oreochromis niloticus, tilapia). This complementary DNA codes for a protein of 248 amino acids, including a putative selenocysteine (Sec) residue, encoded by a TGA triplet, at position 126. The 3' untranslated region contains two putative Sec insertion sequence (SECIS) elements. Recombinant enzyme expressed in COS-1 cells catalyzes both ORD of T4 and rT3 and IRD of T3 and T3 sulfate with the same substrate specificity as native tilapia D1 (tD1), i.e. rT3 >> T4 > T3 sulfate > T3. Native and recombinant tD1 show equally low sensitivities to inhibition by PTU, iodoacetate, and gold thioglucose compared with the potent inhibitions observed with mammalian D1s. Because the residue 2 positions downstream from Sec is Pro in tD1 and in all (PTU-insensitive) type II and type III iodothyronine deiodinases but Ser in all PTU-sensitive D1s, we prepared the Pro128Ser mutant of tD1. The mutant enzyme showed strongly decreased ORD and somewhat increased IRD activity, but was still insensitive to PTU. These results provide new information about the structure-activity relationship of D1 concerning two characteristic properties, i.e. catalysis of both ORD and IRD, and inhibition by PTU

Fish and amphibians as test organisms for evaluation of effects caused by chemicals

A large number of chemicals can contaminate aquatic environments and therefore be exposed to fish and amphibians during their sensitive stages of development. This raises the need for robust methods to identify chemicals that disturb the developmental process. In this thesis, methods for toxicity testing and biomonitoring were developed for zebrafish (Danio rerio) and West-African clawed frog (Xenopus tropicalis). Using these methods, two groups of substances that have achieved attention during recent years were tested, synthetic musks and brominated flame retardants, as well as substances with known mechanism of action. Moreover, zebrafish embryos were used to evaluate chemically complex extracts prepared of effluent water from oil/gas production platforms. Exposure was performed on the embryo stages, to reveal embryo toxic endpoints and in connection to the metamorphosis process in frogs, to evaluate disturbances of the thyroid hormone system. Both methods were able to detect adverse effects in exposed animals. The studies showed that some musk substances had toxic effects on embryos in environmentally relevant concentrations. Embryo toxic responses of musk ketone (MK) and tetrabromobisphenol-A (TBBPA) were recorded in zebrafish as well as in Xenopus tropicalis and moor frog (Rana arvalis) at comparable concentrations. Zebrafish embryos were adequate for monitoring the toxic impact of effluent water from oil/gas production platforms. Effects on X. tropicalis tadpoles due to exposure to propylthiouracil were reduced development and decreased hind limb length, which can be explained by thyroid disruption. Increased sensitivity of the method was achieved by measurements on histological preparations of the thyroid glands. Exposure to polybrominated diphenylethers resulted in signs of thyroid disrupting properties of one tested congener, BDE-99. Moreover, distribution of BDE-99 in tadpole and juvenile X. tropicalis showed long-term retention and accumulation in adipose tissue