Comparison of the effects of 3,5,3'-triiodothyroacetic Acid and triiodothyronine on goiter prevention and involution and on hepatic and skeletal parameters in rats.

3,5,3'-triiodothyroacetic acid (TRIAC) has been used to suppress pituitary TSH secretion with reported attenuation of extrapituitary effects. We investigated whether equivalent doses of T (3) and TRIAC preventing the induction of goiter by methimazole (MMI) had a different or similar impact on peripheral tissues, such as liver and bone. In particular, we compared the effects of both compounds on the activity of the hepatic thyroid hormone-responsive enzymes, malic enzyme and L-glicerol-3-P dehydrogenase; bone mineral density and biochemical parameters of bone turnover, such as bone alkaline phosphatase (b-ALP) and the carboxy-terminal telopeptide region of type I collagen (beta-CTX); and the activity of thyroid ornithine decarboxylase (ODC). We also compared the effects of T (3) and TRIAC on the involution of MMI-induced goiter. Our results showed that TRIAC was more effective than T (3) to reduce MMI-induced goiter in a short-term goiter involution assay. TRIAC increased hepatic enzymes activity and beta-CTX levels, a parameter of bone resorption, more than T (3). However, bone mineral density was not altered by either treatment. Both compounds even reduced ODC activity at doses that were not effective at the pituitary level. These results demonstrate increased TRIAC hepatic and antigoitrogenic activity compared to T (3). TRIAC induces an imbalance in bone remodeling without affecting bone mineral density. Further studies are required to clarify this point.

Hexachlorobenzene, a dioxin-type compound, increases malic enzyme gene transcription through a mechanism involving the thyroid hormone response element.

Hexachlorobenzene (HCB) is a dioxin-type chemical that acts mainly through the aryl hydrocarbon receptor. Chronic exposure of rats to HCB increases the activity of malic enzyme (ME). In this report, we show that this increase is correlated with an induction of ME messenger RNA (mRNA) levels, with the maximal HCB effect achieved after 9 days of intoxication. This effect is specific for ME, as other liver enzymes, such as glyceraldehyde-3-phosphate dehydrogenase, phosphoenol pyruvate carboxykinase, and mitochondrial alpha-glycerol-3-phosphate dehydrogenase, are not affected by HCB. The induction of ME mRNA levels is accompanied by an increase in ME promoter activity, as demonstrated by transient transfection experiments performed in rat hepatoma H35 cells. In an attempt to identify the cis-regulatory elements responsible for the HCB effect, different promoter deletions and mutations were used. The results obtained localize the responsive region between positions -315 and -177. This region does not contain either consensus xenobiotic response or activating protein-1 elements, the two main mediators of dioxin compounds described to date. In contrast, a thyroid hormone response element (TRE) is located between -281 to -261. Deletions and mutations of the TRE element do not respond to HCB, demonstrating that this element mediates the response of this dioxin-type compound. As ME gene expression is regulated mainly by thyroid hormones, we next investigated the role of T3 receptor (T3R) in the ME gene transcriptional induction mediated by HCB. Using Scatchard analysis, we show that neither T3R binding features for its ligand nor alpha1 or beta1T3R mRNA levels are changed with the toxic. In gel shift assays, however, we observed that protein/DNA complexes formed on TRE from the ME promoter were induced by HCB. Using an oligonucleotide with a mutation that eliminates the TRE function, we demonstrate a loss of the induced protein/DNA complexes. Together, these data suggest that the dioxin-type compound HCB increases ME gene transcription by modulating the levels of still unidentified nuclear proteins that bind to the TRE element of the ME promoter.

[Effect of thyroid hormones on the modulation of genetic expression of liver cytosolic malic enzyme, in rats poisoned with hexachlorobenzene]. / Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno.

Hexachlorobenzene (HCB) is a widespread environmental pollutant. Chronic exposure of laboratory animals to HCB triggers porphyria, induction of liver microsomal enzymes, low levels of T4 reproductive dysfunction's, liver and thyroid tumors. Previous findings from our laboratory have shown that HCB increased the activity of the liver thyroid-responsive enzymes: malic enzyme (ME), glucose-6-phosphate dehydrogenase (G6PD) without any change in the mytochondrial alpha-glycerophosphate dehydrogenase (alpha-GPD). In this study we have demonstrated that HCB treatment increased ME mRNA. We also have investigated if HCB affected: a) the thyroid hormone receptor (TR) concentration and binding affinity for its ligands, b) specifically the ME gene expression, or other thyroid hormone responsive enzymes were affected as well, c) Protein/DNA complex formed on the thyroid responsive element (TRE). Livers from female Wistar rats intoxicated with HCB (100 mg/100 g b.w.), for 9 and 15 days, were analyzed. Northern blot hybridization analysis, have demonstrated that ME mRNA levels increased 4 times and 2 times after 9 and 15 days intoxication respectively, without any alterations in the mRNA levels of other thyroid hormone responsive enzymes such as glyceraldheyde 3- phosphate dehydrogenase, phosphoenolpyruvatecarboxikinase and alpha-GPD. These results suggest that HCB affects specifically, ME gene expression. Hepatic T3 and T4 levels evaluated by RIA were not affected by HCB. Scatchard analyses showed that TR affinity and number of sites were not altered after 9 and 15 days of HCB treatment (control, Ka: 1.9 nM, Bmax 3.9 f/mol 100 micrograms DNA: HCD 9 days Ka: 2.1 nM, Bmax 4.5 fmol/100 micrograms DNA: HCB 15 days Ka 1.9 nM. Bmax 5.1 fmol/100 micrograms DNA intoxication, neither at 9 nor at 15 days. Electrophoresis mobility shift assay showed that HCB did not modify nuclear protein extract affinity for the TREs sequence. Our results suggest that TR itself was not directly involved in the induction of ME gene expression by HCB. Nevertheless TR could interact with other transcription factors in the overexpression of ME gene.

Hexachlorobenzene-induced alterations of rat hepatic microsomal membrane function.

The time and dose-dependent effects of the in vivo administration of hexachlorobenzene (HCB), on hepatic microsomal membrane functions, were studied in female Wistar rats. Administration of HCB (100 mg/100 g b.w.) resulted in time-dependent decreases in the activity of two membrane-bound enzymes: 5'nucleotidase and Na+/K+ ATPase. HCB was found to cause a significant rise in protein tyrosine kinase (PTK) activity during the early stages of intoxication (day 2), followed by a significant decrease at 10 days, returning to control levels after 20 days of treatment. A stimulatory effect of HCB on in vitro endogenous microsomal protein phosphorylation was observed from 2 days of intoxication up to 30 days of treatment, with an important stimulation of phosphorylation at 5 days. Administration of HCB (100 mg/100 g b.w.) for 10 days caused a 50% reduction in epidermal growth factor receptor (EGF-R) ligand binding. The effects of known specific inhibitors of protein phosphatases on endogenous protein phosphorylation were studied. HCB affected the labelling of several bands, as well as the 5'nucleotidase and PTK activities, in a dose-dependent manner. In conclusion, this study indicated that the in vivo administration of HCB results in a significant alteration of membrane function.

Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno / Action of the thyroid hormone on modulation of the gene expression of the liver cytosolic malic enzyme, in intoxicated rats with hexachlorobenzene

El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM.

Acción de las hormonas tiroideas sobre la modulación de la expresión genética de la enzima málica citosólica hepática, en ratas intoxicadas con hexaclorobenceno / Action of the thyroid hormone on modulation of the gene expression of the liver cytosolic malic enzyme, in intoxicated rats with hexachlorobenzene

El hexaclorobenceno (HCB) es un tóxico ampliamente distribuído en la biosfera. La exposición crónica de animales de laboratorio al HCB provoca disfunciones tiroideas. Previamente hemos demostrado que el HCB incrementa la actividad de enzimas hepáticas reguladas por hormonas tiroideas (HT) tales como: enzima málica (EM) y glucosa-6fosfato de dehidrogenasa (G6PD) sin alterar la actividad de la alpha-glicerol fosfato deshidrogenasa mitocondrial (alpha-GPD). En éste estudio hemos investigado si el HCB afectaba: a) la concentración del receptor de hormonas tiroideas (RT3) y su afinidad por el ligando, b) la expresión del gen de EM y de otras enzimas HT-dependientes, c) los complejos proteína/DNA formados sobre el elemento de respuesta a hormonas tiroideas (TRE). Se utilizaron hígados de ratas hembras Wistar intoxicadas con HCB (100 mg/100 g P.C.), por 9 y 15 días. El análisis de Scatchard mostró que ni la afinidad ni el número de sitios RT3 estaban alterados luego de 9 y 15 días de tratamiento con HCB (Control, Ka: 1,9 nM, Bmáx:3.9 fmol/100mug DNA; HCB9díasKa2.1nM, Bmáx4.5 fmol/100mug DNA; HCB15 días Ka 1.9nM, Bmáx5.1 fmol/100mug DNA). Tampoco los niveles de RNAm de TRbeta1 medidos por ensayos de protección a RNasa fueron afectados por HCB. Ensayos de Northern Blot han demostrado que los niveles de RNAm de EM se incrementaban 4 veces y 2 veces con respecto al control después de 9 y 15 días de intoxicación respectivamente, sin observarse alteraciones en los niveles de RNAm de otras enzimas cuya expresión es regulada por HT como gliceraldehído - 3 - fosfato deshidrogenasa (GAPDH) y fosfoenolpiruvatocarboxiquinasa (PEPCK) ni tampoco en la alpha-GPD mitocondrial. Ensayos de retardo en gel mostraron que el HCB no modificó la afinidad de las proteínas presentes en extractos nucleares por el TRE presente en el promotor de EM. Nuestros resultados sugieren que el RT3 no está involucrado en forma directa en la inducción de la expresión del gen de EM por HCB, sin embargo podría interaccionar con otros factores de transcripción en la sobreexpresión del gen de EM. (AU)