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. / Graduation date: 1997
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/34437 |
| Date | 08 April 1997 |
| Creators | Sparrow, Barney R. |
| Contributors | Schaup, Henry W. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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