help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Loaiza-Pérez, A. I.
Right arrow Articles by Santisteban, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Loaiza-Pérez, A. I.
Right arrow Articles by Santisteban, P.
Endocrinology Vol. 140, No. 9 4142-4151
Copyright © 1999 by The Endocrine Society

ARTICLES

Hexachlorobenzene, a Dioxin-Type Compound, Increases Malic Enzyme Gene Transcription through a Mechanism Involving the Thyroid Hormone Response Element1

Andrea I. Loaiza-Pérez, Maria-Teresa Seisdedos, Diana L. Kleiman de Pisarev, Horacio A. Sancovich, Andrea S. Randi, Ana M. Ferramola de Sancovich and Pilar Santisteban

Instituto de Investigaciones Biomédicas Alberto Sols, Consejo Superior de Investigaciones Científicas y Universidad Autónoma de Madrid (A.I.L.-P., M.-T.S., P.S.), 28029 Madrid, Spain; Departamento de Bioquímica Humana, Facultad de Medicina (D.L.K.P., A.S.R.), and Departamento de Química Biologica, Facultad de Ciencias, Universidad de Buenos Aires (A.I.L., H.A.S., A.M.F.S.), 1121 Buenos Aires, Argentina

Address all correspondence and requests for reprints to: Pilar Santisteban Ph.D., Instituto de Investigaciones Biomédicas (CSIC-UAM), Arturo Duperier #4, 28029 Madrid, Spain.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 {alpha}1 or ß1T3R 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
HEXACHLOROBENZENE (HCB) is one of the most widespread environmental pollutants known. It was formerly used as a fungicide, and it is currently released as a product derived from the synthesis of other polyhalogenated compounds (1). Chronic exposure of laboratory animals to HCB elicits a number of effects, such as triggering porphyria (2, 3), induction of liver microsomal enzymes (4), reduced levels of serum T4 (3, 5), reproductive dysfunction (6, 7), immunosupression (8), and liver and thyroid carcinogenesis (9, 10). HCB can be classified by its mode of action as a dioxin-type chemical (11). Almost all of the toxic effects of this kind of compounds are due to their interaction with the aryl hydrocarbon receptor (AhR) (12), which has been described as a ligand-dependent transcription factor that belongs to the basic-helix-loop-helix superfamily of DNA-binding proteins. It has been proposed that these compounds enter into the cell through the plasma membrane and bind the Ah-receptor complex, consisting of heat shock proteins (90, 70, and 50), an Src-protein kinase, and AhR (13, 14). After binding, the AhR leaves the complex and moves into the nucleus, where it forms a heterodimer with the Ah receptor nuclear translocator protein (15) and modulates the expression of genes that have specific xenobiotic- or dioxin-responsive elements (XRE or DRE) in their promoters (16). In addition, the protein kinase of the AhR complex can initiate a phosphorylation cascade. In the case of 2,3,7,8-tetrachloro-dibenzo-p-dioxin (TCDD), phosphorylation of epidermal growth factor receptor, induction of the Ras/mitogen-activating protein kinase pathway, and activation of the activating protein-1 (AP-1) complex has been described. This second pathway does not require any ligand-induced receptor interaction with XRE sequences (17).

Taking into account that dioxin-type chemicals modify the expression of a number of metabolic genes, it is of interest to investigate the putative interference between the HCB signaling pathway and the transcriptional machinery that modulates the expression of those genes. A previous study has demonstrated that HCB increases the activity of some thyroid-responsive lipogenic enzymes in liver, such as cytosolic malic enzyme (ME: EC.1.1.1.40), glucose-6-phosphate dehydrogenase (EC.1.1.1.49) and 6-phosphogluconate dehydrogenase (EC.1.1.1.44), with no change in the activity of the mitochondrial {alpha}-glycerol-3-phosphate dehydrogenase ({alpha}GPD; EC 1.1.99.5) (18). These researchers have also shown that the induction of these enzymes was dependent on thyroid hormone (TH) levels.

In this work, we have focused our attention on ME. The results show that ME messenger RNA (mRNA) levels increase 4-fold in the liver of rats intoxicated for 9 days with HCB. The compound seems to induce specifically the expression of the cytosolic ME gene and not other TH-responsive genes, such as cytosolic glyceraldehyde phosphate dehydrogenase (GAPDH; EC 1.2.1.12) (19), phosphoenol pyruvate carboxykinase (PEPCK; EC 4.1.1.32) (20), or mitochondrial {alpha}GPD (21). The effect takes place at the transcription level, as ME promoter activity increased after its transfection into H35 hepatoma cells treated with HCB. To understand the molecular mechanisms involved in the transcriptional control of the ME gene by HCB, different deletions and mutations of the promoter were used. The results obtained demonstrate that no consensus xenobiotic elements (XRE) (16) are present in the ME promoter and that a construct containing the AP-1 element (22, 23) does not respond to HCB. Interestingly, we demonstrate that the TH response element (TRE) located between -281 to -261 (24), is the cis-element responsible for the HCB effect. ME gene expression is regulated mainly by TH (24) acting through the TRE present in the ME promoter (25). This fact and the above-mentioned observation that HCB induces ME activity only in the presence of TH (18) led us to hypothesize that the TH receptor could be the putative transcription factor directly affected by HCB and responsible for ME mRNA induction. HCB could also affect the receptor binding parameters for its ligand. The results obtained here indicate that HCB does not induce changes either in T3 receptor (T3R) binding characteristics for its ligand or in {alpha}1 or ß1 TH mRNA levels. Using the -281 to -261 TRE of the ME promoter, however, we found a clear induction of protein/DNA complexes using liver nuclear extracts from rats treated for 9 days with HCB. Similar results were obtained in H35 cells treated with 50 nM HCB for 48 h. Competition experiments demonstrate that the complexes are specific, and by using a mutated TRE we directly implicate this element in the induction observed. Although the nature of these nuclear proteins remains to be elucidated, our results describe for the first time the induction of ME gene transcription by a dioxin-type chemical, and this effect involves the TRE cis-element of the ME promoter, but not the {alpha}1 or ß1 receptor. However, we cannot exclude that other nuclear receptors and/or associated proteins could be induced in response to HCB and will be the final inductors of ME gene transcription.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Female Wistar rats (160–180 g at the start of the experiment) were maintained in environmental conditions consisting of a 12-h light, 12-h dark cycle at 22 C. The NIH Guidelines for Human Care and Use of Laboratory Animals were followed. HCB (1 g/kg BW) was administered daily through a stomach tube. The fungicide (40 mg/ml) was dissolved in water containing Tween-20 (0.5 ml/100 ml). Control animals received equal volumes of the appropriate vehicle (Tween-20). The dose used elicited clear manifestations of hepatic porphyria, as previously reported (18). Animals were killed by decapitation; the livers were rapidly removed, immediately frozen in dry ice, and stored at -80 C. HCB for rat treatment was of commercial grade (95% pure) and was a gift from Compañía Química S.A. (Buenos Aires, Argentina).

Cell culture
H35 rat hepatoma cells (H4IIE; ATCC CRL 1548, American Type Culture Collection, Manassas, VA) were cultured in DMEM with 5% FCS, 5% newborn calf serum, 100 µg/ml streptomycin, 100 IU/ml penicillin, nonessential amino acids, and sodium pyruvate. The HCB used in cell culture was chemical grade (99% pure; Sigma Chemical Co., St. Louis, MO); it was dissolved in ethanol and used at a final concentration of 10 or 50 nM.

Parameters of thyroid function
T4 and T3 levels were measured in hepatic tissue by specific, highly sensitive RIA as described by Morreale de Escobar et al. (26). In brief, methanol was added to the still frozen tissue samples and homogenized, with tracer amounts of [131I]T4 and [125I]T3 added to each homogenate. This was followed by the addition of chloroform, in a volume double that of methanol, centrifugation, and a further extraction of the pellet with chloroform-methanol (2:1). This extracts more than 90% of the endogenous and added iodothyronines. The iodothyronines are then back-extracted into an aqueous phase and purified by passing this phase through a Bio-Rad Laboratories, Inc. AG 1x2 resin columns (Bio-Rad Laboratories, Inc., Richmond, CA). After performing a pH gradient, the iodothyronines are eluted with 70% acetic acid, evaporated to dryness, and dissolved in RIA buffer. Each extract is extensively counted to determine the recovery of the tracer added to each sample in the initial homogenate. The samples are subjected to highly sensitive RIAs for the determination of T4 and T3; the limits of detection are 2.5 pg for T4 and 1.5 pg for T3/tube. Each sample is processed in triplicate at two or more dilutions.

For Scatchard analysis, liver nuclei were prepared as previously described (27). The nuclear pellet was resuspended in 5 ml SMT buffer (0.32 M sucrose, 1 mM MgCl2, and 20 mM Tris, pH 7.8) containing 2 mM dithiothreitol. The DNA content was determined by the method of Labarca and Paigen (28). The samples values were interpolated in a standard curve of absorbance vs. concentration. The maximum binding capacity and the association constant (Ka) of T3 binding to liver nuclei were determined by Scatchard plot analysis. Nuclei were incubated in SMT buffer with increasing concentrations of [125I]T3 ranging from 7.8 x 10-11 to 2.5 x 10-9 M. A parallel set of tubes was prepared to determine nonspecific binding as follows. Nuclei were incubated with the same amount of [125I]T3 plus a saturating nonradioactive T3 concentration (2.9 x 10-7 M). All determinations were performed in duplicate. After incubation (135 min, 20 C), the nuclei were cooled to O C and centrifuged. The nuclear pellet was washed twice in SMCT containing 0.5% Triton X-100, and bound radioactivity was determined in a {gamma}-counter. Specific binding was calculated by subtracting nonspecific binding from total binding in each tube.

RNA extraction and Northern blot analysis
Livers from control and treated rats were homogenized in guanidinium isothiocyanate, and total RNA was extracted after centrifugation through a cesium chloride cushion (29). Total RNA from cells was extracted as described previously (30). Polyadenylated [poly(A)+] RNA was obtained using the mRNA separator kit (CLONTECH Laboratories, Inc., Palo Alto, CA). Samples of total RNA or poly(A)+ were electrophoresed in 1% agarose gels containing 2.2 M formaldehyde (31), blotted on Nytran membranes (Schleicher & Schuell, Inc., Keene, NH) and hybridized using the following rat probes: ME complementary DNA (cDNA) (32), GAPDH (33), PEPCK (34), {alpha}GPD (35), and ß-actin (36). The ME probe was prepared by digesting the plasmid pMEB, which contains full-length ME cDNA, with BamHI and BglII. The GAPDH insert subcloned in pGEM plasmid was obtained by digestion with PstI. PEPCK cDNA subcloned in pPCK10 was obtained after digestion with PstI. The {alpha}GPD insert subcloned in pBluescript IISK was obtained by digestion with EcoRI. All probes were isolated and labeled by random oligopriming to a specific activity of 1 x 109 cpm/DNA. When total RNA was used, the filters were stained with methylene blue to correct for the amount of applied RNA. In the case of poly(A)+, filters were hybridized with the ß-actin probe. The levels of mRNA were determined by autoradiography and quantitated by densitometric scanning. The results were expressed as an intensity percentage compared with the control expression of each experiment.

Ribonuclease (RNase) protection assay
A 265-bp XbaI/EcoRI restriction fragment of the c-erbA (37) (TR{alpha}1) probe or the 232-bp PstI-BamHI restriction fragment of the rat T3 receptor ß (38) (TRß1) probe was cloned in the pBS+ SK vector as described previously (39). 32P-Labeled antisense riboprobes were generated using T7 RNA polymerase, [{alpha}-32P]CTP (800 Ci/mmol; Amersham Pharmacia Biotech, Aylesbury, UK) and the Riboprobe Transcription Kit (Promega Corp., Madison, WI) according to the manufacturer’s instructions. As an internal control to correct for total RNA loading, an additional 28S ribosomal antisense riboprobe was transcribed from pT7RNA-28S. The mRNA levels were analyzed by RNase protection with the RPA II assay kit (RPA II RNase protection assay kit, Ambion, Inc. Austin, TX), essentially as described by the manufacturer. TR{alpha}1 and TRß1 (5 x 105 cpm) and 5 x 104 cpm 28S riboprobes were incubated overnight with 20 µg total RNA. After hybridization, samples were digested at 37 C with a RNase mixture (2.5 U/ml RNase A and 50 U/ml T1 RNase) for 30 min for TR{alpha}1 and 2 h for TRß1 and 28S. The protected fragments were precipitated and electrophoresed on a denaturing 5% acrylamide gel, dried, and visualized by autoradiography. The TR{alpha}1 and TRß1 mRNA levels and ribosomal 28S RNA were quantified by densitometric gel scanning in an Instantimager (Packard, Meridien, CT), and the relative TR{alpha}1 and TRß1 mRNA levels were expressed in arbitrary units after correction with 28S ribosomal RNA levels.

Transfection assays
5'-Deletions of the rat ME promoter [pME 882-chloramphenicol acetyltransferase (CAT), pME 315-CAT, and pME 177-CAT] (22) and the mutated constructs, pME 882/{Delta}Cla-CAT and pME 882/m277-CAT (40), were used to study ME promoter activity. Point mutagenesis was generated by Desvergne et al. (40) using the oligonucleotide-directed mutagenesis system (Amersham Pharmacia Biotech) to create ClaI sites at positions -260 and -298 of the ME gene. Oligonucleotides encompassing the ME gene sequences from -240/-274 and -285/-315 with different mutations, extensively described previously (40), were annealed to M13 gene fragment of pME882-CAT. After the synthesis, the mutated fragment was inserted in the KpnI/PstI site of pUCATSV I (22) generating pME 882/ClaI-CAT. An internal deletion was then obtained by ClaI digestion followed by ligation to generate pME-882/{Delta}ClaI-CAT. The mutation pME 882/m277-CAT was generated by a point mutation introduced at -277 (A->C).

Ten micrograms of each construct were transiently transfected by the calcium phosphate method (41) together with 2 µg of a plasmid directing luciferase expression from the cytomegalovirus (CMV) promoter (42) to correct for transfection efficiency. After transfection, half of the plates were treated with 50 nM HCB for 48 h, cell extracts were prepared, and CAT as well as luciferase activities were assayed as previously described (43, 44).

Preparation of nuclear extracts and gel shift assay
Nuclear extracts were prepared following the method of Gorski et al. (45) for liver and the method of Andrew and Faller (46) for cells. Proteins were determined by the Bradford method (47) using the Bio-Rad kit (Bio-Rad Laboratories, Inc.) with a BSA standard. Gel shift assays were performed with oligonucleotides labeled with T4 polynucleotide kinase and [{gamma}-32P]ATP and annealed as previously described (48). For binding reactions, 5 or 10 µg nuclear protein from cells or rat liver, respectively, were preincubated in a binding reaction mixture containing 40 mM HEPES (pH 7.9), 200 mM KCl, 0.5 mM dithiothreitol, 0.2 mM EDTA, 5% Ficoll, and 3 µg poly(dI-dC) for 15 min on ice. In competition experiments, the unlabeled competitor oligonucleotide was added in excess (100-fold). Then, 50 pg labeled oligonucleotide were added to the mixture and incubated for 30 min at room temperature. The resulting DNA-protein complexes were separated from free DNA on a 5% polyacrylamide gel (29:1, acrylamide-bisacrylamide). Gels were resolved at 20 mA in a cold room in 0.5 x TBE (1 x TBE is 90 mM Tris, 90 mM boric acid, and 1 mM EDTA, pH 8) before being vacuum dried and exposed to x-ray film at -70 C.

Statistical analysis
The mRNA levels and the promoter activity results were expressed as an intensity percentage compared with the control expression of each experiment. Each value represents the mean ± SD of three different experiments. The values from RIA T3 and T4 as well as from the Scatchard analysis are the mean ± SD. Statistical significance between the control and the different treatments was determined by Student’s t test. Differences are considered significant at P < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Hexachlorobenzene is a potent inductor of ME gene expression in liver and does not affect other TH-responsive genes
We first studied whether the reported increase in ME activity due to HCB (18) correlates with an increase in ME mRNA levels. Rats were intoxicated daily by gastric intubation with HCB for 9 or 15 days. These intoxication periods were chosen because ME activity reached 75% of its maximum value after 9 days of treatment with HCB (18) and were extended to 15 days when other manifestations of hepatic intoxication become evident (49). The percentage of ME activity is referred to as a maximum determined after 4 weeks of treatment in previous studies (18). After death, livers were excised, and total RNA was extracted and analyzed by Northern blot. Treatment with HCB for 9 days strongly increased ME mRNA levels (4-fold over control values; Fig. 1Go). After 15 days of treatment, a 2-fold increase in ME mRNA compared with the control value was observed. As ME is a cytosolic lipogenic enzyme mainly regulated by TH (24), we next investigated whether HCB affected the expression of other cytosolic thyroid-responsive enzymes involved in different metabolic pathways. We analyzed the HCB effect on GAPDH, a glucolytic enzyme, and on PEPCK, a key gluconeogenetic enzyme. We performed Northern blot analysis using total RNA from liver of control and intoxicated rats. HCB did not alter either GAPDH or PEPCK mRNA levels (Fig. 1Go).



View larger version (37K):
[in this window]
[in a new window]
  		Figure 1. Effect of HCB on TH-responsive liver enzyme gene expression. mRNA levels of cytosolic ME, GAPDH, and PEPCK and mitochondrial {alpha}GPD were measured in rat liver after HCB treatment (1 g/kg BW). The pesticide was administered to rats for 9 or 15 days. Total RNA (30 µg) was used to determine ME, GAPDH, and PEPCK transcripts, whereas 5 µg poly(A)+ were used for {alpha}GPD mRNA levels. A, Upper panel, A representative Northern blot of each experimental group. The sizes of the mRNAs are indicated. A, Lower panel, The methylene blue staining of membranes with total RNA in which the positions of ribosomal RNAs are indicated. Membranes with poly(A)+ were hybridized with ß-actin. B, Quantification of mRNAs, after correction with 28S or ß-actin mRNA, by densitometer scanning of autoradiograms from three independent experiments. The data are the mean ± SD of three independent experiments. In the case of ME, control levels can be detected and measured after longer exposures.

 
We also studied the effect of HCB on the mRNA levels of a mitochondrial TH-responsive enzyme, {alpha}GPD. As mRNA levels of liver {alpha}GPD are low, we used poly(A)+ mRNA instead of total RNA to perform Northern blot analysis. No changes in {alpha}GPD mRNA levels were observed after treatment with HCB (Fig. 1Go), confirming a previous report that showed no effect of HCB on {alpha}GPD activity (18). Our results demonstrate that the HCB effect is specific for the ME gene and that the toxic does not have a generalized action on the expression of other cytosolic or mitochondrial TH-dependent enzymes.

HCB stimulates ME promoter activity through the TRE element
To determine whether the HCB-induced increase in ME mRNA levels was due to an increase in ME gene transcription, we followed a transfection experimental approach using H35 hepatoma cells. As in rat liver, these cells respond to HCB by increasing the ME mRNA levels after 48 h of treatment in a dose-dependent manner (Fig. 2Go). The construct pME 882-CAT (23, 40), which contains the full-length ME promoter (Fig. 3AGo), was transiently transfected into H35 cells. Half of the cell plates were then treated for 48 h with 50 nM HCB, a concentration that showed maximal ME mRNA induction in Northern blot experiments (Fig. 2Go, line 3). ME promoter activity was determined by assaying crude cell extracts for CAT activity, whereas luciferase activity derived from the cotransfected CMV-Luc was used to correct for variability in transfection efficiency. The results obtained demonstrate that HCB increases ME promoter activity 3- to 4-fold (Fig. 3BGo), indicating that its effect on ME gene expression takes place at the transcriptional level. To define the region within the promoter responsible for the HCB response, several 5'-deletions (23, 40) of the ME promoter (Fig. 3AGo) were transfected as described above. As shown in Fig. 3BGo, the deletion pME-315 responds to HCB as the full promoter does, whereas no response was found with the deletion pME-177. This data suggest that cis-acting elements that respond to HCB should be located in a region between -177 to -315 of the ME promoter. To identify this cis-regulatory element, we first analyzed by computer whether any of the previously described response elements for dioxin-type compounds (DRE/XRE) were present between -177 to -315. No consensus DRE or XRE were found in this region. However, a previously described functional TRE is located within this region (from -261 to -281) (25). To assess whether the TRE element is involved in the HCB response, two constructs containing, respectively, a deletion of the entire TRE (pME882/{Delta}ClaI) or a punctual mutation on nucleotide 277 (pME-882/m277) together with the full promoter with an additional ClaI site (Fig. 3CGo) were transfected into H35 cells treated or not with 50 nM HCB. Again, the full promoter with the ClaI site responds 3- to 4-fold to HCB, as the ClaI mutation does not modify the activity of the promoter (40). The response was totally lost when the TRE element was either deleted or mutated (Fig. 3DGo). These data strongly suggest that the TRE of the ME promoter is involved in the HCB response.



View larger version (53K):
[in this window]
[in a new window]
  		Figure 2. HCB increases ME mRNA levels in H35 cells. A representative Northern blot containing 30 µg total RNA from H35 control cells or treated for 48 h with 10 or 50 nM HCB and hybridized with the ME probe (upper) or stained with methylene blue (lower).

 


View larger version (27K):
[in this window]
[in a new window]
  		Figure 3. HCB effect on ME promoter activity and identification of the HCB responsive region. A and C, Schematic diagram of the full ME promoter (pME-882), the different deletions (pME-315 and pME-177), and the deleted (pME-882/{Delta}ClaI) or mutated (pME-882/m277) promoters generated over the pME-882ClaI construct linked to the CAT reporter gene. Binding sites detected previously are indicated with the corresponding names of the elements. B and D, ME promoter activity derived from 10 µg of each construct transiently transfected into H35 cells. After transfection, half of the plates received 50 nM HCB. Forty-eight hours later, the cells were harvested, and CAT and luciferase activities were determined. The ME promoter activity (CAT/luciferase) is expressed as the fold induction over the control levels (=1) of untreated cells. The results are the mean ± SD of four independent experiments.

 
Moreover, this transfection analysis also demonstrates that the AP-1 element of the ME promoter (22, 23) located at -132 bp (Fig. 3AGo), which has been described to mediate the dioxin-response in other genes (17), is not involved in the HCB regulation of ME gene promoter, as the construct pME-177 that contains this element does not respond to HCB. In addition, no changes were observed with the toxic in either the AP-1/DNA complex or the levels of c-Fos and c-Jun proteins (data not shown).

Parameters of thyroid function in HCB-intoxicated rats
As ME gene transcription is directly controlled by TH (24) and the levels of serum TH have been described to be altered in the plasma of HCB-treated rats (18), our next aim was to determine whether HCB induces ME gene transcription through the TRE present in the ME promoter. We first analyzed the thyroid status of the intoxicated animals. Previous data have shown that serum T4 levels are reduced after 9 and 15 days of HCB intoxication, whereas T3 levels are not significantly altered (50, 51). Using sensitive RIA assays, we measured hepatic T4 and T3 levels after HCB treatment. In contrast with the data on serum TH levels, we found that hepatic tissue levels of T4 are not affected by the pesticide, whereas T3 levels are slightly reduced at both times of treatment (Table 1Go). We next examined whether HCB affects T3 binding affinity for its receptor or induces changes in the number of sites. Nuclei from control and intoxicated rats were isolated, and Scatchard plot analysis was performed. Table 2Go shows that neither 9 nor 15 days of HCB treatment affected the kinetic parameters of T3R.


View this table:
[in this window]
[in a new window]
  		Table 1. Influence of HCB on T4 and T3 levels in rat liver

 

View this table:
[in this window]
[in a new window]
  		Table 2. [125I]T3 binding receptor parameters

 
TR ({alpha}1 and ß1) mRNA levels are unaltered by HCB treatment
Most parameters of thyroid status studied to date were unaltered or showed only very small changes to explain the increase found in ME gene expression after HCB treatment. TR is the primary transcription factor that mediates the action of TH. There are various isoforms of TRs produced from two different genes, {alpha} and ß (52). TR{alpha}1 and TRß1 are able to bind to ME TRE (25). Therefore, another parameter potentially responsible for the increase in ME gene expression is a change in the expression levels of TRs. We studied the mRNA levels of TR{alpha}1 and TRß1 after HCB treatment. Total RNA from the livers of each experimental group was isolated, and TR{alpha}1 and ß1 mRNA levels were determined by RNase protection assay. The TR{alpha}1 probe is 328 nucleotides (nt) long and contains, in addition to 63 nt of vector sequences, 265 nt complementary to TR{alpha}1 mRNA (39). The TRß1 probe is 263 nt long and contains, in addition to 31 nt of vector sequence, 232 nt complementary to TRß1 (39). A major 265- or 232-nt-long protected RNA fragment was detected, respectively, for TR{alpha}1 or TRß1 in livers of control rats and in 9- and 15-day HCB-treated rats (Fig. 4Go, A and B). Other fragments of smaller size were also detected, presumably originating from spurious cleavage by RNase. The most abundant 265- and 232-nt fragments were used in densitometric analysis, as we observed that all fragments are identically regulated. Scanning densitometry of three separate experiments showed the same levels of TR{alpha}1 and TRß1 mRNA in all groups (Fig. 4CGo), corroborating the lack of HCB effect observed on T3R concentration by Scatchard analysis (number of sites; Table 2Go).



View larger version (52K):
[in this window]
[in a new window]
  		Figure 4. TH receptor (TR {alpha}1 and ß1) mRNA levels in rat liver in response to HCB. HCB (1 g/kg BW) was administered to rats for 9 or 15 days, and total liver RNA was extracted. RNase protection assays showing the 265-nt fragment of TR {alpha}1 mRNA (A) or showing in duplicate the 232-nt fragment of the TRß1 mRNA (B) are represented. In both cases, the 115-nt fragment of 28S ribosomal RNA and the riboprobes with or without RNase are shown. C, Quantification of mRNAs by densitometric gel scanning. Each value represents the mean ± SD of three independent experiments. For quantification of 28S in the experiments with TR{alpha}1, the gel scanning was measured after less exposure.

 
HCB increases protein/DNA complexes formed on TRE from the ME promoter
As TR ({alpha}1 and ß1) mRNA levels do not change after HCB treatment, we determined whether the toxic affects the binding of other nuclear proteins to the TRE from the ME promoter. We carried out gel shift assays with nuclear extracts from livers of control and 9- and 15-day HCB-treated rats and the oligonucleotide (5'-GTTGGGGTTAGGGGAGGACAG-3), corresponding to the TRE element from the ME gene promoter (-281 to -261; Fig. 5AGo). Three protein/DNA complexes were found in nuclear extracts of control rat livers (lane 2), although the two top complexes (1, 2) were almost undetectable. Interestingly HCB treatment for 9 days induced the binding capacity of the three complexes, but this induction was much more pronounced in the top complexes 1 and 2 (lane 3). Treatment with HCB for 15 days did not modify the intensity of the complexes compared with that in control rats. These three complexes are specific, as they are displaced by a 100-fold excess of unlabeled TRE (lane 5), but not by an unrelated oligonucleotide (lane 6). Similar results were obtained when nuclear extracts from control or 50 nM HCB-treated H35 cells were used (Fig. 5BGo). To have a more direct demonstration of the TRE implication, we performed gel shift assays with a mutated TRE (5'-GTTGGGGTTAGGGGAGtAaAG'-3). This mutated TRE, in which the -264 G has been replaced by a T and the -262 C has been replaced by an A, virtually eliminates the TRE function, as demonstrated in transfection experiments by Desvergne et al. (40). The results obtained using nuclear extracts from livers of 9-day treated rats (Fig. 5AGo, lane 7) or from 50 nM HCB-treated cells (Fig. 5BGo, lane 4) indicate a loss of binding of all of the induced hepatic DNA-binding activities. These data strongly suggest that still unidentified nuclear proteins that bind to the TRE element of the ME are involved in the HCB-mediated regulation of this gene.



View larger version (70K):
[in this window]
[in a new window]
  		Figure 5. Effect of HCB on protein/DNA complexes formed on TRE-ME. A, Nuclear extracts (10 µg) from livers of control rats (C) or rats treated with HCB for 9 (HCB 9) or 15 days (HCB 15) were incubated with labeled oligonucleotide TRE (-281 to -261 of ME promoter) for 30 min at room temperature. Free and bound DNA were separated as described in Materials and Methods. B, Electrophoretic mobility shift assay performed as described in A with 5 µg nuclear extracts from H35 control (C) or treated for 48 h with 50 nM HCB. For competition, nuclear extracts from livers of rats treated with HCB for 9 days or from cells treated with 50 nM HCB were incubated with a 100-fold excess of unlabeled related (TRE; A, lane 5, or B, lane 3) or unrelated oligonucleotide (A, lane 6). To study the involvement of TRE, a mutated oligonucleotide TRE mutant 1 (-264 G->T, -262 C->A) were incubated with nuclear extracts from livers of rats treated with HCB for 9 days (A, lane 7) or from H35 cells treated with HCB for 48 h (B, lane 4). The protein/DNA complexes are indicated by arrows.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this paper, we have demonstrated for the first time that HCB, a dioxin-type chemical, is a potent inductor of ME gene transcription. These data are in agreement with previous work showing that HCB increases the activity of various TH-responsive lipogenic enzymes in liver (18) and with other reports that showed the same effect on ME activity for TCDD (53, 54, 55). It has also been shown that HCB, TCDD, and other related dioxin-type compounds decrease plasma T4 levels with no change in T3 levels (49, 53, 54, 55). However, in the present study we demonstrate that HCB does not alter T4 levels and only slightly decreases T3 levels when TH are measured directly in the liver. These differential effects of HCB on TH could be due to the production of some HCB metabolites. In this regard, pentachlorophenol, the main HCB metabolite, competes strongly with T4 for serum carrier proteins (56). This competition could result in an increase in free T4 entering the liver. However, preliminary results from our laboratory indicate that HCB decreases 5-deiodinase activity in liver; consequently, less T4 is converted to rT3. In addition, we observed a decrease in 5'-deiodinase, resulting in less T4/T3 conversion. Whether the effect of HCB on deiodinase activity is a direct effect of HCB or is mediated by its metabolite pentachlorophenol remains to be elucidated. Based on the above explanation it is unlikely that HCB induces a transient hyperthyroidism in liver. Moreover, the discrepant response of hepatic mitochondrial and cytosolic enzymes mRNA levels does not support the hypothesis that HCB-treated rats are hyperthyroid.

In addition, the results obtained suggest that TH availability for TH nuclear receptors is not basically affected by the pesticide. These results are compatible with those reported previously, showing that HCB did not change hepatic tissue thyroid status (18). We have further shown that HCB does not modify the number of T3R sites or its ligand binding affinity, nor are TR{alpha}1 or TRß1 mRNA levels affected. The fact that this compound induces not only ME activity, but also that of other enzymes of the lipogenic pathway, such as glucose-6-phosphate dehydrogenase and 6-phosphate dehydrogenase (18), suggests that NADPH-generating enzymes could be specifically affected by HCB. Taking into account that generation of NADPH enzymes could be involved in detoxification metabolism, their induction by HCB could be viewed as a defensive strategy of animals to the aggression of xenobiotic agents. This is reinforced by the fact that dioxins and other lipophilic xenobiotics produce a rise in other liver detoxification enzymes, such as the cytochrome P450 system (CYP), as well as glucuronyl transferases (57, 58). With respect to the HCB doses used, it is of interest to mention that the dose assayed elicits in rats a specific effect on the ME gene, as the toxic does not affect the expression of other hepatic enzymes, such as the cytosolics GAPDH and PEPCK or the mitochondrial {alpha}GPD. Furthermore, the dose of 50 nM used in H35 cells is on the order of magnitude of the doses used in most of the studies with compounds whose action is mediated by the AhR (59, 60).

The data reported in the present work showing that HCB increases ME promoter activity led us to identify the cis- and trans-regulatory elements involved in the HCB induction of ME gene transcription. The study of the mechanism of action of dioxin-type compounds is becoming increasingly relevant (12, 13). After its interaction with HCB, AhR trans-locates to the nucleus, where the AhR-Ah receptor nuclear translocator heterodimers bind to cis-elements called XRE (16). By computer analyses, no consensus XRE was found in the 882-bp fragment of the ME promoter studied. As the other mechanism of action for dioxin-type compounds is the phosphorylation cascade involving the AP-1 element and c-Fos/c-Jun proteins (17), we investigated the possible involvement of this element in the ME regulation mediated by HCB. The fact that a ME promoter deletion containing the AP-1-binding site does not respond to HCB suggests that this element is not the mediator of the induction found. This result together with the finding of no changes in the levels of AP-1 complexes or in the c-Fos/c-Jun proteins (not shown) are different from those reported by Ashida and Matsumura (61), which showed an increase in the AP-1 complex in response to TCDD as well as an increase in c-Fos and a decrease in c-Jun levels. These differences could be due to the fact that these researchers studied the effect of a different compound (TCDD) in the liver of another species, the guinea pig. The length and mode of intoxication are also totally different in the two experimental situations.

Interestingly, the cis-element involved in the HCB regulation of ME promoter is the TRE, as we have extensively demonstrated in the transfection experiments reported in the present work. The dioxin-type chemicals increase malic enzyme activity only in the presence of TH (18, 53, 54, 55), suggesting that this hormone is a permissive factor for the action of these chemical compounds. ME expression is regulated by nuclear T3 receptor, and although T3R ({alpha}1 and ß1) does not seem to be directly responsible, other transcription factors interacting with the TRE of ME or with the T3R could be involved in the mechanism of action of HCB. In this respect, we found an increase in the protein/TRE-ME complexes when nuclear extracts were used from rat liver treated for 9 days with HCB. At this time of intoxication, there is an increase in all of the complexes (1, 2, 3), although the major increase was observed in complexes 1 and 2. Similar results were obtained in H35 cells. The induction observed in the bottom complex (complex 3) is not constant, suggesting that the main protein/TRE-ME complexes induced in response to HCB are the top complexes 1 and 2. The nature of the protein(s) that forms part of these complexes remain to be elucidated, although they should be related to the TR, because in a gel shift experiment with a mutated TRE that does not respond to TH (40), the binding activity of the three complexes is loss. In addition, preliminary competition experiments using different consensus TREs as competitors suggest that the proteins induced could be members of the nuclear hormone receptor superfamily. However, more experiments are required to definitively establish the identity of such proteins.

T3R, along with receptors for retinoic acid, 9-cis-retinoic acid (RXRs), vitamin D, steroid hormone and peroxisome proliferator activated-receptor (PPAR), belongs to this superfamily of transcription factors (52). T3R binds to TRE as homodimer or heterodimer with RXR. It has been demonstrated that the interaction of RXR-T3R with TRE activates transcription. Taking into account that RXR also forms heterodimers with retinoic acid receptor, vitamin D receptor, PPAR, and orphan receptors, the response of a cell to the TH depends not only on the levels of TH and T3R, but also on the relative RXR and other nuclear receptor levels that compete with T3R to bind with RXR. In future studies, it would be interesting to measure the levels of RXR and other nuclear receptors after HCB treatment. Another possibility that could be considered in future studies is whether coactivators or corepressors of the TRAC family (T3 receptor-associating cofactors) (52) are affected by HCB.

Finally, the HCB-mediated induction of ME promoter activity could also be explained by the fact that this gene is controlled by the peroxisome proliferator-activated receptor that binds to an element (PPRE) located at 338 bp (62). Both AhR and PPAR mediate the induction of different hepatic CYP isoenzymes by their ligands (63). The induction of ME by both receptor-ligand systems provides the NADPH required for the subsequent induction of the CYP activity. However, the fact that the promoter deletion -315 that does not contain the ME-PPRE element still responds to HCB suggests that this element does not mediate the induction observed.

In summary, these data confirm that we have identified the cis-regulatory element that mediates ME gene transcription in response to HCB. In addition, we found that nuclear proteins bound to this element are induced in response to this compound. However, the nature of this trans-acting element still remains to be identified. Future experiments will elucidate the participation of other nuclear receptor or associated proteins in the HCB regulation of ME gene expression.


   Acknowledgments
 
We are especially indebted to Dr. Vera M Nikoden (NIH, Bethesda, MD) for the ME cDNA probes and the ME promoter constructs pME-882 and pME-177, and to Dr. Beatrice Desvergne (Institut de Biologie Animale, Lausanne, Switzerland) for the constructs of deletions and mutations of ME promoter pME-315, pME-ClaI-882, pME-{Delta}Cla, and pME-882m277. We also thank Drs. J. P. García-Ruiz (Centro de Biología Molecular, CSIC-UAM, Madrid, Spain), M. Alexander (Massachusetts General Hospital, Harvard Medical School, Boston, MA), L. J. Brown and M. J. MacDonald (University of Wisconsin, Madison, WI), B. Paterson (NIH), A. Pérez-Castillo (IIB, CSIC-UAM), and A. Santos (Facultad de Medicina, Universidad Complutense, Madrid, Spain) for PEPCK, GAPDH, {alpha}GPD, ß-actin, TRß1, and TR{alpha}1 probes, respectively. We are grateful to Dr. Gabriella Morreale (IIB, CSIC-UAM) for suggestions and for the liver T3 and T4 determinations, and to Dr. Isabel Barroso (IIB, CSIC-UAM) for critical reading of the manuscript.


   Footnotes
 
1 This work was supported by Grants DGICYT (PM97–0065), CAM (08.1/0025/1997), and Fundación Salud 2000 (Spain) and by Grants Universidad de Buenos Aires (ME028) and CONICET (Argentina). Preliminary results have been presented at the 70th Annual Meeting of the American Thyroid Association (Thyroid [Suppl 1] 7:170, 1997) and at the Congress of the Sociedad Argentina de Endocrinología Médica, 1997 (Acta Physiol Pharmacol Ther Lat 48:125, 1998). Back

Received November 23, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Courtney KD 1979 Hexachlorobenzene (HCB): a review. Environ Res 20:225–246[Medline]
  2. Peters HA, Gocman A, Cripps DJ, Bryan GT, Dogramaci Y 1982 Epidemiology of hexaclorobenzene-induced porphyria in Turkey. Arch Neurol 39:744–749[Abstract/Free Full Text]
  3. Rozman K, Gorski JR, Rozman P, Parkinson A 1986 Reduced serum thyroid hormone levels in hexachlorobenzene induced porphyria. Toxicol Lett 30:71[CrossRef][Medline]
  4. Linko P, Yeowell HN, Gasiewicz TA, Goldstein JA 1986 Induction of cytochrome P-450 isoenzymes by hexaclorobenzene in rats and aromatic hydrocarbons (Ah)-responsive mice. J Biochem Toxicol 1:95–107[CrossRef][Medline]
  5. Kleiman de Pisarev DL, Sancovich HA, Ferramola de Sancovich AM 1989 Enhanced thyroxine metabolism in hexachlorobenzene-intoxicated rats. J Endocrinol Invest 12:767–772[Medline]
  6. Foster WG, McMahon A, Villeneuve DC, Jarrell JF 1992 Hexachlorobenzene (HCB) supress circulating progesterone concentrations during the luteal phase in the cynomolgus monkey. J Appl Toxicol 12:13–17[CrossRef][Medline]
  7. Bourque AC, Singh A, Lakhanpal N, McMahon A, Foster WG 1995 Ultrastructural changes in ovarian follicles of monkeys admininistered hexachlorobenzene. Am J Vet Res 56:1673–1677[Medline]
  8. Schielen P, Den Besten C, Vos JG, Van Bladeren PJ, Seinen W, Bloksma N 1995 Immune effects of hexaclorobenzene in the rat: role of metabolism in a 13-week feeding study. Toxicol Appl Pharmacol 131:37–43[CrossRef][Medline]
  9. Cartew P, Smith AG 1994 Pathological mechanism of hepatic tumour formation in rats exposed chronically to dietary hexachlorobenzene. J Appl Toxicol 14:447–452[CrossRef][Medline]
  10. Grimalt JO, Sunyer J, Moreno V, Amaral OC, Sala M, Rosell A, Anto JM, Albeiges J 1994 Risk excess of soft-tissue sarcoma and thyroid cancer in a comunity exposed to airbone organochlorinated compound mixtures with a high hexachlorobenzene content. Int J Cancer 56:200–203[Medline]
  11. Hahn M, Goldstein JA, Linko P, Gasiewicz TA 1989 Interaction of hexachlorobenzene with the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin in vitro and in vivo. Arch Biochem Biophys 270:344–355[CrossRef][Medline]
  12. Whitlock JP Jr 1990 Genetic, and molecular aspects of 2,3,7,8-tetrachloro-dibenzo-p-dioxin action. Annu Rev Pharmacol Toxicol 30:251–257[Medline]
  13. Gonzalez FJ, Liu S-Y, Yano M 1993 Regulation of cytochrome P450 genes: molecular mechanism. Pharmacogenetics 3:213–230[Medline]
  14. Chen H-S, Perdew GH 1994 Subunit composition of the heterodimeric cytosolic aryl hydrocarbon receptor complex. J Biol Chem 269:27554–27558[Abstract/Free Full Text]
  15. Hord NG, Perdew GH 1994 Physicochemical and immunocytochemical analysis of the aryl hygdrocarbon receptor nuclear translocator: characterization of two monoclonal antibodies to the aryl hydrocarbon receptor translocator. Mol Pharmacol 46:618–626[Abstract]
  16. Matsumura F 1995 Mechanism of action of dioxin-type chemicals, pesticides,and other xenobiotics affecting nutritional indexes. Am J Clin Nutr 61:695S–701S
  17. Matsumura F 1994 How important is the protein phosphorylation pathway in the toxic expression of dioxin-type chemicals? Biochem Pharmacol 48:215–224[CrossRef][Medline]
  18. Kleiman de Pisarev DL, Ferramola de Sancovich AM, Sancovich HA 1995 Hepatic indices of thyroid status in rats treated with hexachlorobenzene. J Endocrinol Invest 18:271–276[Medline]
  19. Shimokawa T, Kato M, Shiokuda K, Irie J, Ezaki O 1997 Effect of triiodothyronine on muscle cell differentiation and blood glucose level in hyperglycemic KK mice. Biochem Biophys Res Commun 235:790–793[CrossRef][Medline]
  20. Hanson RW, Reshef L 1997 Regulation of phosphoenolpyruvate carboxykinase (GTP) gene expression. Annu Rev Biochem 66:581–611[CrossRef][Medline]
  21. Wilson EJ, McMurray W C 1981 Regulation of malic enzyme and mitochondrial {alpha}-glycerophosphate dehydrogenase by thyroid hormones, insulin and glucocorticoids in cultured hepatocytes. J Biol Chem 256:11657–11662[Abstract/Free Full Text]
  22. Morioka H, Tennyson GE, Nikodem VM 1988 Structural and functional analysis of the rat malic enzyme gene promoter. Mol Cell Biol 8:3542–3545[Abstract/Free Full Text]
  23. Streeper RS, Chapman SC, Ayala JE, Svitek CA, Goldman JK, Cave A, O’Brien RM 1998 A phorbol ester-insensitive AP-1 motif mediates the stimulatory effect of insulin on rat malic enzyme gene transcription. Mol Endocrinol 12:1778–1791[Abstract/Free Full Text]
  24. Dozin B, Magnuson MA, Nikodem VM 1986 Thyroid hormones regulation of malic enzyme synthesis. J Biol Chem 261:10290–10292[Abstract/Free Full Text]
  25. Petty KJ, Morioka H, Mitsuhashi T, Nikodem VM 1989 Thyroid hormone regulation of transcription factors involved in malic enzyme gene expression. J Biol Chem 264:11483–11490[Abstract/Free Full Text]
  26. Morreale de Escobar G, Calvo R, Escobar del Rey F, Obregón MJ 1994 Thyroid hormones in tissues from fetal and adult rats. Endocrinology 134:2410–2415[Abstract/Free Full Text]
  27. Bernal J, Coleoni AH, DeGroot L 1978 Triiodothyronine stimulation of nuclear protein synthesis. Endocrinology 102:452–459[Abstract/Free Full Text]
  28. Labarca C, Paigen K 1980 A simple rapid and sensitive DNA assay procedure. Anal Biochem 102:344–352[CrossRef][Medline]
  29. Chirgwin M, Przybyla AE, MacDonald RJ, Rutter WJ 1979 Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5300[CrossRef][Medline]
  30. Chomczynski P, Sacchi N 1987 Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  31. Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor
  32. Song M-KH, Dozin B, Grieco D, Rall JE, Nikodem VM 1988 Transcriptional activation and stabilization of malic enzyme mRNA precursor by thyroid hormone. J Biol Chem 263:17970–17974[Abstract/Free Full Text]
  33. Alexander M, Curtis G, Avruch J, Goodman HM 1985 Insulin regulation of protein biosynthesis in differentiated 3T3 adipocytes. J Biol Chem 260:11978–11985[Abstract/Free Full Text]
  34. Yoo Warren H, Monahan JE, Short J, Short H, Bruzel A, Wynshaw-Boris A, Meisner HM, Samols D, Hanson RW 1983 Isolation and characterization of the gene coding for cytosolic phosphoenolpyruvate carboxykinase (GTP) from the rat. Proc Natl Acad Sci USA 80:3656–3660[Abstract/Free Full Text]
  35. Brown LJ, MacDonald MJ, Lehn DA, Moran SM 1994 Sequence of rat mitochondrial glycerol-3-phosphate dehydrogenase cDNA: evidence for EF-hand calcium binding domain. J Biol Chem 269:14363–14366[Abstract/Free Full Text]
  36. Levi A, Eldridge JD, Paterson, BM 1985 Molecular cloning of a gene sequence regulated by nerve growth factor. Science 393–395
  37. Mitsushashi T, Tennyson GE, Nikodem V 1988 Alternative splicing generates messages encoding rat c-erbA protein that do not bind thyroid hormone. Proc Natl Acad Sci USA 85:5804–5808[Abstract/Free Full Text]
  38. Murray MB, Zilz ND, McCreer NL, MacDonald MJ, Towle HC 1988 Isolation and characterization of rat cDNA clones from two distinct thyroid hormone receptors. J Biol Chem 263:12270–12277
  39. Rodriguez-García M, Jolin T, Santos A, Pérez Castillo A 1995 Effect of perinatal hypothyroidism on developmental regulation of rat pituitary growth hormone and thyrotropin genes. Endocrinology 136:4339–4350[Abstract]
  40. Desvergne B, Petty KJ, Nikodem VM 1991 Functional characterization and receptor binding studies of the malic enzyme thyroid hormone response element. J Biol Chem 266:1008–1013[Abstract/Free Full Text]
  41. Chen C, Okayama H 1988 Calcium phosphate-mediated gene transfer: a highly efficient transfection system for stably transforming cells with plasmid DNA. BioTechniques 6:632–639[Medline]
  42. Boshart M, Weber F, Jahn G, Dorsch-Häsler K, Fleckenstein B, Schaffner W 1985 A very strong enhancer is located upstream of a immediate early gene of human cytomegalovirus. Cell 41:3631–3639
  43. Gorman CM, Moffat LF, Howard BH 1982 Recombinanat genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol 2:1044–1051[Abstract/Free Full Text]
  44. de Wet JR, Wood KV, De Luca M, Helinski DR, Subramani S 1987 The firefly luciferase gene: stucture and expression in mammalian cells. Mol Cell Biol 7:725–737[Abstract/Free Full Text]
  45. Gorski K, Carneiro M, Schibler U 1986 Tissue-specic in vitro transcription from the mouse albumin promoter. Cell 47:767–776[CrossRef][Medline]
  46. Andrew NC, Faller DV 1991 A rapid micropreparation technique for extraction of DNA-binding proteins for limiting number of mammalian cells. Nucleic Acids Res 19:249[Abstract/Free Full Text]
  47. Bradford MM 1976 A rapid and sensitive method for the quantification of micrograms quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254[CrossRef][Medline]
  48. Santisteban P, Acebrón A, Polycarpou-Schwartz M, Di Lauro R 1992 Insulin and insulin-like growth factor I regulate a thyroid-specific nuclear protein that binds to the thyroglobulin promoter. Mol Endocrinol 6:1310–1317[Abstract/Free Full Text]
  49. Kleiman de Pisarev DL, Rios de Molina MC, San Martín de Viale LC 1990 Thyroid function and thyroxine metabolism in hexachlorobenzene-induced porphyria. Biochem Pharmacol 39:817–825[CrossRef][Medline]
  50. Sopena de Kracoff YE, Ferramola de Sancovich AM, Sancovich HA, Kleiman de Pisarev D.L 1994 Effect of thyrodectomy and thyroxine on hexachlorobenzene induced porphyria. J Endocrinol Invest 17:301–305[Medline]
  51. Van Raaij JAGM, Frijters CMG, Van Der Berg KJ 1993 Hexachlorobenzene induced hypothyroidism involvement of different mechanism by parent compound and metabolite. Biochem Pharmacol 46:1385–1391[CrossRef][Medline]
  52. Lazar MA 1993 Thyroid hormone receptors: multiple forms, multiple possibilities. Endocr Rev 14:184–193[Abstract/Free Full Text]
  53. Roth W, Voorman R, Aust SD 1988 Activity of thyroid-hormone-inducible enzymes following treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Toxicol Appl Pharmacol 92:65–74[CrossRef][Medline]
  54. Kelling CK, Menahan LA, Peterson RE 1987 Hepatic indices of thyroid status in rats treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem Pharmacol 36:283–291[CrossRef][Medline]
  55. Schuur AG, Boekhorst FM, Bouwer A, Visser TJ 1997 Extrathyroidal effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on thyroid hormone turnover in male Sprague-Dawley rats. Endocrinology 138:3727–3734[Abstract/Free Full Text]
  56. Van Raaij JAGM, Van Den Berg KJ, Notten WR 1991 Hexachlorobenzene and its metabolites pentachlorophenol and tetrachlorohydroquinone: interaction with thyroxine binding sites of rat thyroid hormone carriers ex vivo and in vitro. Toxicol Lett 59:101–107[CrossRef][Medline]
  57. Hankinson, O 1995 The aryl hydrocarbon receptor complex. Annu Rev Pharmacol Toxicol 35:307–340[CrossRef][Medline]
  58. Sutter TR, Greenlee WF 1992 Classification of members of the Ah gene battery. Chemosphere 25:223–226[CrossRef]
  59. McNully SE, Toscano WA 1995 Transcriptional regulation of glyceraldehyde-3-phosphate dehydrogenase by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Biochem Biophys Res Commun 212:165–171[CrossRef][Medline]
  60. Backlund M, Johansson I, Mkrtchian S, Ingelman-Sundberg M 1997 Signal transduction-mediated activation of the aryl hydrocarbon receptor in rat hepatoma H4IIE cells. J Biol Chem 272:31755–31736[Abstract/Free Full Text]
  61. Ashida H, Matsumura F 1998 Effect of in vivo administered 2,3,7,8-tetrachlorodibenzeno-p-dioxin on DNA-binding activities of nuclear transcription factors in liver of guinea pigs. J Biochem Mol Toxicol 12:191–204[CrossRef][Medline]
  62. Castelein H, Gulick T, Declercq PE, Mannaerts GP, Moore DD, Baes M 1994 The peroxisome proliferator activated receptor regulates malic enzyme gene expression. J Biol Chem 269:26754–26758[Abstract/Free Full Text]
  63. Gonzalez FJ, Fernández-Salguero P, Lee SS, Pineau T, Ward JM 1995 Xenobiotic receptor knockout mice. Toxicol Lett 83:117–112



This article has been cited by other articles:


Home page
 International Journal of ToxicologyHome page
Y. E. Sopena, A. M. Ferramola de Sancovich, and H. A. Sancovich
Hexachlorobenzene Treatment on Hepatic Mitochondrial Function Parameters and Intracellular Coproporphyrinogen Oxidase Location
International Journal of Toxicology, November 1, 2008; 27(6): 455 - 465.
[Abstract] [Full Text] [PDF]

Home page
 Eur J EndocrinolHome page
M. Boas, U. Feldt-Rasmussen, N. E Skakkebaek, and K. M Main
Environmental chemicals and thyroid function.
Eur. J. Endocrinol., May 1, 2006; 154(5): 599 - 611.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Loaiza-Pérez, A. I.
Right arrow Articles by Santisteban, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Loaiza-Pérez, A. I.
Right arrow Articles by Santisteban, P.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals