Baculovirus vectors repress phenobarbital-mediated gene induction and stimulate cytokine expression in primary cultures of rat hepatocytes.

Baculovirus transfection strategies have proven successful at transferring foreign DNA into hepatoma cells and primary hepatocytes. When testing the utility of these methodologies in cultured hepatocytes, we discovered that the presence of baculovirus disrupts the phenobarbital (PB) gene induction process, a potent transcriptional activation event characteristic of highly differentiated hepatocytes, and repressed expression of the albumin gene. In concert with previous reports from our laboratory demonstrating that increased cAMP levels can completely repress the induction of specific cytochrome P450 (CYP) genes, cAMP concentrations and PKA activities were measured in the primary hepatocytes subsequent to baculovirus exposure. However, neither parameter was affected by the presence of the virus. To evaluate whether immune response modulation was triggered by baculovirus exposure, RNase protection assays were performed and demonstrated that baculovirus infection activates TNF-alpha, IL-1alpha and IL-1beta expression in the primary hepatocyte cultures. Immunocytochemical experiments indicated that the production of cytokines was likely due to the presence of small numbers of Kupffer cells present in the culture populations. Exogenously added TNF-alpha was also effective in repressing PB induction, consistent with other reports indicating that inflammatory cytokines are capable of suppressing expression of biotransformation enzyme systems. Comparative studies demonstrated the specificity of these effects since exposures of hepatocytes to adenoviral vectors did not result in down-regulation of hepatic gene responsiveness. These results indicate that baculovirus vectors enhance the expression of inflammatory cytokines in primary hepatocyte cultures, raising concerns as to whether these properties will compromise the use of baculovirus vectors for study of cytochrome P450 gene regulation, as well as for liver-directed gene therapy in humans.

Lack of modulation by phenobarbital of cyclic AMP levels or protein kinase A activity in rat primary hepatocytes.

Results of previous studies have substantiated a negative modulatory role for cyclic AMP (cAMP) and protein kinase A (PKA) dependent processes on the phenobarbital (PB) induction response in hepatocytes. The current study was conducted to further examine the potential role of second messenger pathways in the initial phases of induction, specifically addressing the effects of PB on the expression of intracellular cAMP levels and associated PKA activity. Using a highly differentiated primary rat hepatocyte system, cells were exposed to PB for various intervals (30 sec to 48 hr), and levels of intracellular cAMP and subsequent PKA activity were determined. Although PB markedly induced CYP2B expression, exposure to this agent produced no detectable increases in cAMP levels and PKA activity at any of the times examined. These results demonstrated that the initial events stimulated by PB in rat hepatocytes do not include alterations of cAMP levels or associated PKA activities.

Phenobarbital responsiveness conferred by the 5'-flanking region of the rat CYP2B2 gene in transgenic mice.

Phenobarbital (PB) is a prototype for a class of agents that produce marked transcriptional activation of a number of genes, including certain cytochrome P-450s. We used transgenic mouse approaches and multiple gene reporters to assess the functional consequences of specific deletions and site-specific mutations within the 2.5kb 5'-flanking region of the rat CYP2B2 gene. Protein-DNA interactions at the PBRU domain also were characterized. Using the transgenic models, we demonstrate that sequences between -2500 and -1700bp of the CYP2B2 gene are critical for PB induction; mice with 1700 or 800bp of 5'-flanking CYP2B2 sequence are not PB responsive. DNA affinity enrichment techniques and immunoblotting and electromobility shift assays were used to determine that nuclear factor 1 (NF-1) interacts strongly with a site centered at -2200bp in the PB responsive unit (PBRU) of CYP2B2. To test the functional contribution of NF-1 in PB activation, we introduced specific mutations within the PBRU NF-1 element and demonstrated that these mutations completely ablate the binding interaction. However, transgenic mice incorporating the mutant NF-1 sequence within an otherwise wild-type -2500/CYP2B2 transgene maintained full PB responsiveness. These results indicate that, despite the avidity of the respective DNA-protein interaction within the PBRU in vitro, NF-1 interaction is not an essential factor directing PB transcriptional activation in vivo.

The human microsomal epoxide hydrolase gene (EPHX1): complete nucleotide sequence and structural characterization.

Human microsomal epoxide hydrolase (mEH) is a xenobiotic-metabolizing enzyme that detoxifies reactive epoxides to more water soluble dihydrodiol compounds. We have isolated and sequenced clones that encode the entire human mEH gene (EPHX1). The primary nuclear transcript, extending from the start of transcription to the site of poly(A) addition, is 20,271 nucleotides in length. The human mEH gene contains 9 exons, separated by 8 introns; canonical intron/exon boundary sites are observed at each junction. The introns vary in size from 335 to 6696 bp and contain numerous repetitive DNA elements, including 18 Alu sequences (each > 100 nucleotides in length) within 4 introns. Alu sequences were classified with respect to subfamily assignment. Two thousand eighteen nucleotides 5' of the transcription start and 2501 nucleotides 3' of the poly(A) addition sites were also sequenced. To evaluate the human mEH promoter, chimeric constructs were prepared linking portions of the 5' mEH flanking sequence (up to -693 bp) to a CAT reporter gene, followed by transient transfection in both COS-1. and HepG2 cells. Results from these expression experiments suggest that the human mEH gene contains a weak core promoter and that inclusion of DNA sequences 5' of the minimal promoter region negatively regulates constitutive transcription.