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

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.

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 -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 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.

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