Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced modification of hepatic pyruvate carboxylase gene expression in C57BL/6J male mice

Abstract

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure on hepatic pyruvate carboxylase (PC) gene expression were investigated in C57BL/6J Ah[superscipt b/b male mice. A dose-dependent reduction of PC levels and activity occurred in animals given a single intraperitoneal dose of TCDD in a corn oil carrier. The dose ranged from 1 to 75 ug/kg body weight and the analysis performed 8 days postinjection. At the maximum TCDD level investigated, a 10-fold reduction in PC activity occurred. At doses beyond those required to initiate a reduction in PC, a lactate dehydrogenase isozyme patterns shift is observed. This is accompanied by increases in blood lactic acid levels. Northern blot analysis on RNA extracts from hepatic tissues indicated that at 8 days post TCDD treatment, a dose-dependent reduction of hepatic mRNA levels occurs. The aryl hydrocarbon receptor (AhR) is believed to mediate all responses to TCDD. Liganded AhR and the aryl hydrocarbon receptor nuclear translocator (ARNT) protein form a heterodimeric transcription factor which interacts with dioxin response elements (DREs). These are found in enhancer/promoter regions of many genes that respond transcriptionally to TCDD exposure. Cloning and sequencing a region approximately 1.4 kb upstream of the PC translational start site revealed an untranslated leader sequence of 124 nucleotides starting with adenosine. Primary structural analysis of the upstream region revealed an 1nr element in place of a TATA element. Additional transcription factor elements were identified including: Spl, GCF, UBP-1, GRE, CREB, NF-1, HNF-4, TFII-I and E-boxes; DRE elements were notably lacking. A tandem series of 10 evenly spaced E-boxes, which bind ARNT homodimers, are each juxtaposed to a TFII-I element, possibly forming composite elements. Tertiary structure analysis revealed the positioning of nine composite elements displayed as a trio of phased elements. Transient transfections into Hepa lc1c7 cells, using a luciferase reporter gene under the transcriptional control of the PC upstream region, unlike the animal studies, produced an induction in activity in the presence of 10 nM TCDD. Co-transfections with an ARNT encoding plasmid reduced induction indicating overexpression of ARNT protein partially overrides the TCDD-induced increase in activity. These results in relationship to whole animal experiments are discussed