Glucuronidation of thyroid hormone in rat liver: effects of in vivo treatment with microsomal enzyme inducers and in vitro assay conditions.

We investigated the effects of in vivo treatment with different microsomal enzyme inducers, including clofibrate (CLOF), hexachlorobenzene (HCB), 3-methylcholanthrene (MC), 3,3',4,4'-tetrachlorobiphenyl (TCB), and 2,3,7,8-tetrachloro-p-dioxin, as well as of in vitro addition of the detergent Brij 56 on the glucuronidation of T4, T3, and rT3 by UDP-glucuronyltransferase (UGT) activities of rat liver microsomes. The results were compared with measurements of UGT activities for bilirubin, p-nitrophenol (PNP), and androsterone. In general, glucuronidation rates were 5-fold or more higher with rT3 than with T4 or T3 as substrate. In liver microsomes from untreated rats, T4 UGT activity was stimulated by Brij 56 to a maximum of about 2-fold at 0.025% detergent. Treatment of Wistar rats for 4 days with CLOF (200 mg/kg BW.day) resulted in significant increases in UGT activities for T4 (to 154%), rT3 (to 155%), and bilirubin (to 194%), in particular if assayed in the presence of 0.025% Brij 56, but had little effect on the UGT activities for T3, PNP, and androsterone. The CLOF-induced increases in T4 and rT3 UGT activities were not observed in Gunn rats, which have a complete lack of bilirubin UGT activity and greatly impaired PNP UGT activity. Treatment of Wistar rats with a single injection of MC (50 mg/kg BW), TCB (50 mg/kg BW), or 2,3,7,8-tetrachloro-p-dioxin (6.25 micrograms/kg BW) resulted, after 4 days, in 6.3- to 7.3-fold increases in T4 UGT activity and 15.1- to 16.7-fold increases in rT3 UGT activity if determined in the absence of Brij 56, whereas T4 UGT activity was only increased by 33-68% when assayed in the presence of Brij 56. T3 glucuronidation was not affected (with Brij 56) or was increased by only 33-68% (without Brij 56) after treatment with these MC-type inducers. PNP UGT activity was induced 3.6- to 4.3-fold, whereas bilirubin and androsterone UGT activities were changed little by these treatments. Similar findings regarding T4, rT3, PNP, and bilirubin UGT activities were obtained after chronic treatment of WAG rats with HCB, another MC-type inducer. However, WAG rats lack androsterone UGT and show low T3 UGT activity, which was increased about 2.3-fold by HCB treatment. On the basis of these and previous findings it is concluded that at least three UGT isoenzymes are involved in the glucuronidation of thyroid hormone.(ABSTRACT TRUNCATED AT 400 WORDS)

Multiple UDP-glucuronyltransferases for the glucuronidation of thyroid hormone with preference for 3,3',5'-triiodothyronine (reverse T3).

We have studied the glucuronidation of the thyroid hormones T4, T3 and rT3 by liver microsomes of Wistar, Gunn and WAG rats. Gunn rats have a defect in the gene coding for bilirubin and phenol UDP-glucuronyltransferase (UGT) isoenzymes; WAG rats have a genetic defect in androsterone UGT. In normal Wistar rats UGT activity was approximately 5-fold higher for rT3 than for T4 or T3. UGT activities for T4 and rT3, but not for T3, were impaired in Gunn rats. Conversely, UGT activity for T3, but not for T4 or rT3, was impaired in WAG rats. Thus, in rat liver rT3 is glucuronidated much more rapidly than T4 and T3. Our results support the view that T4 and rT3 are glucuronidated by bilirubin and phenol UGTs and T3 by androsterone UGT.

Increased glucuronidation of thyroid hormone in hexachlorobenzene-treated rats.

Metabolism of thyroid hormones was investigated in WAG/MBL rats that had been exposed to hexachlorobenzene (HCB). Serum thyroxine (T4) levels were lowered by 35.5%, whereas triiodothyronine (T3) levels were not changed. Bile flow, as well as T4 excretion in bile were increased by HCB treatment. Analysis of bile by HPLC revealed a more than 3-fold increase of T4 glucuronide (T4G) and a concomitant reduction of non-conjugated T4. T4 UDP-glucuronyltransferase activity (T4 UDPGT) activity in hepatic microsomes was increased more than 4.5-fold in animals exposed to HCB. p-Nitrophenol (PNP) UDPGT showed a comparable increase by HCB. Both T3 and androsterone UDPGT activities were low in WAG/MBL rats compared with normal Wistar rats. T3 UDPGT activity was increased 2.5-fold by HCB, but androsterone UDPGT activity was unchanged. These results suggest that T4 is a substrate for HCB-inducible PNP UDPGT and T3 for androsterone UDPGT. In the absence of the latter, T3 is also glucuronidated to some extent by PNP UDPGT. Type 1 iodothyronine deiodinase activity was decreased by HCB treatment. It is concluded that decreased T4 levels in serum of animals after exposure to HCB may be due to a combined effect of displacement of T4 from carriers, an increased glucuronidation of T4 and enhanced bile flow.

Hexachlorobenzene-induced hypothyroidism. Involvement of different mechanisms by parent compound and metabolite.

Rats received repeated oral treatment with different doses of hexachlorobenzene (HCB) (0-3.5 mmol/kg) for 2 or 4 weeks. Measurements of thyroid hormone status after 2 weeks showed a dose-dependent decrease of total thyroxine (TT4) levels, decreased free thyroxine (FT4) levels and little change of total triiodothyronine (TT3) levels. The effects on thyroid hormone status were more pronounced after 4 weeks and also included increased thyroid stimulating hormone (TSH) levels. These conditions suggest that HCB had induced hypothyroidism in these animals. Indications for occupation of thyroid hormone binding proteins were found in serum of exposed animals. The major metabolite pentachlorophenol (PCP) also caused, by competitive interactions with thyroid hormone binding proteins in serum, a rapid and dose-dependent decrease of TT4 and FT4 levels, but not of TT3 levels in serum. The decrease of serum TT4 levels by repeated dosing with 3.5 mmol HCB/kg for 4 weeks could be attributed to competitive interactions of PCP with hormone serum binding proteins and to increased metabolism induced by HCB to an equal degree. At lower dose levels or with shorter dosing periods, increased metabolism of T4 is the main cause of decreased TT4 serum levels. This is the first indication that a similar effect is caused simultaneously by the parent compound and its metabolite through different and independent mechanisms.

Effects of hexachlorobenzene and its metabolites pentachlorophenol and tetrachlorohydroquinone on serum thyroid hormone levels in rats.

Effects of administration of equimolar doses of hexachlorobenzene (HCB) and its metabolites pentachlorophenol (PCP) and tetrachlorohydroquinone (TCHQ) on serum thyroxine (TT4) and triiodothyronine (TT3) levels in rats were studied. Furthermore, it was investigated whether the observed effects were related to the serum levels of HCB or PCP. Rats received either corn oil (controls) or HCB, PCP or TCHQ in a single equimolar intraperitoneal dose of 0.056 mmol/kg. Results indicated that HCB did not alter serum TT4 and TT3 levels for a period up to 96 h after dosing. In contrast, PCP and TCHQ were both capable of reducing serum TT4 levels with a maximum effect between 6 and 24 h after exposure. TCHQ was more effective in repressing TT3 than TT4 blood levels. Dose-response experiments were carried out in order to obtain insight into the sensitivity of the observed effects. Rats received different doses of PCP or TCHQ intraperitoneally. The reductions of TT4 levels by PCP were inversely related to serum PCP levels in exposed animals, based on the toxicokinetics and dose-response profiles. Furthermore, PCP serum levels after HCB administration appeared too low to cause an effect. The results of this study indicate that not HCB itself, but rather its metabolites PCP and TCHQ may be involved in reduced serum thyroid hormone levels after HCB administration.

Reduction of thyroxine uptake into cerebrospinal fluid and rat brain by hexachlorobenzene and pentachlorophenol.

In the present study the effects of hexachlorobenzene (HCB) and the metabolite pentachlorophenol (PCP) were investigated with respect to uptake of thyroxine (T4) into cerebrospinal fluid (CSF) and brain structures of rats. [125I]T4 was taken up into CSF of control rats by a relatively slow process, reaching a steady state after about 3 h. Both repeated dosing of HCB and single doses of PCP caused decreased uptake of [125I]T4 into CSF, total brain tissue as well as specific brain structures, such as occipital cortex, thalamus, and hippocampus. Although HCB-treatment caused a build-up of HCB and PCP levels in serum in brain only HCB was present in significant amounts (16% of the serum level). In CSF, both HCB and PCP concentrations were below detection levels. Separate experiments with PCP showed, however, a dose- and time-dependent uptake of PCP into CSF. The present results indicate that PCP and the parent compound HCB are able to affect brain supply of T4. This may have consequences for an adequate development of the brain or proper brain function in adults. The exact mechanisms of interference of PCP and/or HCB in brain uptake of T4 remain to be established.

Hexachlorobenzene and its metabolites pentachlorophenol and tetrachlorohydroquinone: interaction with thyroxine binding sites of rat thyroid hormone carriers ex vivo and in vitro.

Previous results have indicated that hexachlorobenzene (HCB)-induced hypothyroidism may be caused by its main metabolite pentachlorophenol (PCP), and by tetrachlorohydroquinone (TCHQ), rather than by the parent compound. In the present experiments it was investigated whether hormone displacement from serum carriers could be a factor in the development of this hypothyroidism. In an in vitro competition assay PCP was an effective competitor for the thyroxine (T4)-binding sites of serum carriers, whereas HCB was ineffective. Ex vivo experimental results demonstrated occupation of T4-binding sites in sera from PCP-exposed animals but not in sera from HCB- or TCHQ-treated animals. Competing ability for T4-binding sites was still present in sera of PCP-exposed animals but was absent in HCB- or TCHQ-exposed animals. The results suggest that thyroid hormone displacement by the major metabolite PCP may play a role in HCB-induced hypothyroidism.

Quantitative analysis of computer-averaged electromyographic profiles of intrinsic limb muscles in ponies at the walk.

The function of several intrinsic muscles of the fore-and hind limbs of 5 ponies walking normally was evaluated via surface electromyography. Electromyographic signals were band-pass filtered, rectified, linear enveloped, and standardized to the stride duration. Mean data from the muscles of the left and right limbs that were obtained from at least 30 strides in 2 recording sessions were recorded as electromyographic signals-time curves. The timing of muscle activity was determined from these graphs. On the basis of the major peaks in the electromyographic signal, muscle functions were identified. In the forelimb, the extensor carpi radialis muscle was involved in extension of the carpus at the end of the swing phase of the stride, and it provided support to flexion of the cubital joint at the beginning of the swing phase. The common digital extensor muscle extended the distal joints of the forelimb at the end of the swing phase. The ulnaris lateralis muscle provided support to extension of the cubital joint at the beginning of the stance phase, and the flexor carpi radialis muscle flexed the carpus at the beginning of the swing phase. The flexor carpi ulnaris muscle extended the cubital joint at the end of the swing phase. In the hind limb, the long digital extensor muscle flexed the tarsus at the beginning of the swing phase and extended the digital joints preceding the stance phase. The deep digital flexor muscle prevented overextension of the distal interphalangeal joint during the stance phase and flexion of the digital joints during the swing phase.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions of halogenated industrial chemicals with transthyretin and effects on thyroid hormone levels in vivo.

Previous results in experimental systems have suggested that hydroxylated PCBs may decrease thyroid hormone levels through associative interaction with transthyretin. In the present paper it was investigated whether this property was also shared by various industrial chemicals, mainly pesticides. In total, 65 compounds from 12 chemical groups were analyzed for direct interference with the T4 binding site of transthyretin using a competitive binding assay. Sixty per cent of the compounds were competitive at a concentration level of 100 microM. Relatively strong interactions were observed by several chlorophenols, chlorophenoxy acids and nitrophenols, as well as by individual compounds such as hexachlorobenzene, dicofol, bromoxynil and tetrachlorohydroquinone. Examples from these chemical groups, e.g. pentachlorophenol, 2,4-dichlorophenoxybutyric acid, dinoseb and bromoxynil, also reduced plasma TT4 levels in rats. In addition, bromoxynil decreased plasma TT3 levels. The results suggest the existence of a number of halogenated industrial chemicals with a potential for lowering plasma thyroid hormone levels through interference with hormone transport carriers.