Date on Master's Thesis/Doctoral Dissertation


Document Type

Doctoral Dissertation

Degree Name

Ph. D.


Biochemistry and Molecular Biology

Committee Chair

Prough, Russell A.

Author's Keywords

Aldehyde dehydrogenase; Activation protein 1 (AP-1); Acrolein; 4-hydroxy-2-nonenal; Lipid peroxidation; Toxicity




The lipid aldehydes, 4-hydroxy-2-nonenal (4-HNE) and propene-2-al (acrolein) are reactive a,ß-unsaturated aldehydes generated during the peroxidation of lipids and are implicated in the pathogenesis of several oxidative-stress mediated diseases, including steatohepatitis and cancer. We established that mouse liver aldehyde dehydrogenase 1 al (Aldhlal) efficiently metabolizes lipid aldehydes and protects a liver-derived cell line from the toxic effects of these aldehydes. Thus, mechanisms to induce the expression of Aldhlal might be a useful rationale for preventing oxidative stress-induced pathologies. We investigate whether well-known electrophiles, such as BHA or acrolein modulate the expression of Aldhlal and elucidate the signaling pathway involved. Microarray analyses were performed to examine whether acrolein or BHA up-regulate the expression of genes encoding enzymes involved in antioxidant or electrophile detoxification in mice liver. Mice were administered AIN76A (control) diet, diet containing 0.45% BHA or 5 mg/kg acrolein by gavage for 7 days. The expression of genes encoding several electrophile detoxifying enzymes was specifically elevated, indicating a detoxification response. The elevation of Aldhlal was noticeable, with a 2- to 3-fold increase by both electrophiles. Quantitative real-time PCR analysis also showed ˜2.5-fold and ˜3- fold induction of Aldhlal gene expression by BHA and acrolein, respectively. Livers from BHA- and acrolein-treated mice also showed increased cytosolic Aldh activity compared to control. Acrolein and tert-butylhydroquinone (the metabolized products of BHA) are electrophiles that induce the expression of cytoprotective genes by direct activation of nuclear factor-E2-related factor-2 (Nrf2), activator protein 1 (AP-l) and nuclear factor kappa B (NF-?B) transcription factors or indirectly by activation of protein kinases, such as MAPKs. To decipher the signaling pathways involved in A1dhlal induction by electrophiles, we analyzed the mRNA levels of Aldhlal in the liver of Nrf2+I+ and Nrf2-/- mice on C57BL6 background exposed to BRA. Mice exposed to BRA showed ˜2-fold increase in mRNA levels of Aldhlal in both Nrf2+I+ and Nrf2-/-mice compared to control, indicating that electrophile-induced expression might be independent ofNrf2. However, the mRNA and protein levels of AP-l and the activity of c-Jun were significantly increased by BRA. We hypothesized that electrophile-induced expression of hepatic Aldhlal gene is mediated by activation of AP-1 transcription factor. Transient transfection experiments were conducted in HepG2 cells with Aldhlal 5'-flanking luciferase reporter constructs. While co-transfection with Nrf2 expression plasmid alone or in the presence of tBRQ had no effect, over-expression of c-Jun/AP-l resulted in ˜4-fold induction in Aldhlal transcriptional activity. Moreover, c-Jun transactivates Aldhlal promoter activity as a homodimer and not c-Jun/c-fos heterodimer. We also established by promoter deletion and mutagenesis analysis that two AP-l sites at position -758 and -1069 relative to Aldhlal transcription start site are responsible for c-Jun-mediated transactivation of Aldhlal luciferase activity. EMSA analysis using biotin- labeled probe and super shift with antibodies against c-Jun, c-fos and Nrf2 showed that c-Jun binds to the proximal AP-1 site at -758 but not at -1069. The recruitment of c-Jun to this AP-1 site by BHA was confirmed by ChIP experiment, which showed ˜10-fold enrichment to the proximal AP-1 site with c-Jun. These results indicate that electrophiles promote the recruitment of c-Jun/AP-1 to the Aldh1a1 gene promoter, resulting in increased transcription of Aldh1al.