A number of xenobiotic toxins alter expression of a subset of specific genes in the liver by first inducing expression of the gene encoding the transcription factor, ATF3, which is a bZIP (basic region-leucine zipper) protein and a member of the ATF/CREB family of transcription factors. Consensus sequences for ATF3 binding have been identified in a number of liver-specific gene promoters, specifically those thought to regulate ornithine transcarbamylase, aldolase, transferrin, alpha-antitrypsin, apolipoprotein B, some acute phase genes, e.g., glutathione-S-transferase, and specific cytochrome P450 genes. ATF3 is induced by physiological stressors including: a) exposure to agents such as carbon tetrachloride, acetaminophen and alcohol, b) mechanical injury, c) restriction of blood flow to the heart, and d) brain seizure. Its expression correlates with cellular injury and cell death. Therefore, this project focuses on the role of ATF3 in xenobiotic-induced liver toxicity in humans. The principal investigator hypothesizes that """"""""perturbation of the bZip pool will lead to the de-regulation of liver functions."""""""" Thus, the goal of this proposal is to test the hypothesis by over-expressing ATF3 in the liver using a tissue-specific binary transactivator/transresponder transgenic mouse model.
The specific aims are to: 1) create individual transactivator and transresponder transgenic mice, 2) to cross the lines to create the biogenic mice, and 3) to test the consequences of """"""""deregulated over-expression"""""""" of ATF3 in adult animals and during embryonic development. A liver-specific, transthyretin-driven """"""""reverse tetracycline"""""""" induction system will be used to regulate hepatic expression of an ATF3 transgene (""""""""tagged"""""""" with a hemagglutinin [HA] sequence that is detectable by immunological methods using antiHA antibodies). To achieve this, lines of low-copy transgenic mice expressing transthyretin-directed tetracycline repressor constructs (""""""""transactivators"""""""") will be bred to lines of low-copy transgenic mice expressing the ATF3-HA fusion construct (""""""""transresponders"""""""") to create biogenic or double-transgenic lines. Offspring will be used to study perturbation of the ATF3 system during adulthood as well as during the period of fetal (hepatic) development by xenobiotic agents. ATF-transgene expression will be examined in adult transgenic mice. The question to be addressed is whether increased ATF3 expression increases the sensitivity to xenobiotic agents. Sub-optimal concentrations of xenobiotic agents will be tested to see if heightened ATF3 expression in biogenic mice potentiates effects of these agents. The effects of aberrant ATF-transgene expression on liver function will also be examined during embryonic development.
