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DIR Papers of the Month

By Eddy Ball
December 2006

Network Analysis in Toxicology Studies

An NIEHS-funded research team has demonstrated the utility of systematic statistical analysis of microarray data for identifying differences between gene expression levels and patterns in high and low doses of acetaminophen. The team included investigators from the Laboratory of Molecular Toxicology and the Environmental Toxicology Program working in collaboration with Japanese researchers.

The team investigated gene interaction networks of 17 genes from livers of rats orally exposed to 50, 150 and 1500 mg/kg acetaminophen at 6, 24 and 48 hours after exposure. Using a variety of statistical and bioinformatics approaches, the researchers clustered nine dose-time observation points. Their networks related genes associated with oxidative stress to genes associated with apoptosis and demonstrate markedly different networks for the two lowest doses relative to the highest dose.

The results of this study indicate that gene interaction network analysis may prove useful for the development of biomarkers and assessing chemical toxicity. The gene interaction network/clustering approaches demonstrated in this study can address questions of dose-dependent toxic responses seen in pharmaceutical applications and answer them in a scientifically rigorous manner.

Citation: Toyoshiba H, Sone H, Yamanaka T, Parham FM, Irwin RD, Boorman GA, Portier CJ. 2006. Gene interaction network analysis suggests differences between high and low doses of acetaminophen. Toxicol Appl Pharmacol 215(3):306-316.

P-glycoprotein Up-regulation/Reduced Penetration of Therapeutic Drugs into CNS

Researchers from the NIEHS Laboratory of Pharmacology and Chemistry, in collaboration with UNC pharmacologists, have demonstrated the role of a nuclear receptor, the human pregnane-X receptor (hPXR), in altering the efficacy of a CNS-acting drug. The NIEHS-funded study showed that up-regulation of the blood-brain barrier transport protein P-glycoprotein by rifampin, an antibiotic and hPXR ligand, reduced the analgesic effects of methadone.

The experiments were performed in vivo and in vitro using male CB6F1 wild type mice and CB6F1 transgenic mice expressing hPXR. Researchers determined that pretreatment with rifampin reduced the pain-relieving effects of methadone in test animals by 70%, even though blood levels of the CNS-acting drug in test animals were similar to levels in controls not given the antibiotic.

Since the transgenic mice exhibited plasma rifampin levels identical to those found in patients, the findings suggest that hPXR-based changes in selective barrier function are likely to be widespread in the human population and may affect drug efficacy and safety. Blood-brain barrier P-glycoprotein is a major impediment to pharmacotherapy for a number of CNS diseases, including brain cancer and epilepsy. Alterations in transporter activity may underlie the multi-drug resistance seen in 30 % of epilepsy patients as well as patient-to-patient variability in response to CNS-acting drugs.

Citation: Bauer B, Yang X, Hartz AM, Olson ER, Zhao R, Kalvass JC, Pollack GM, Miller DS. 2006. in vivo activation of human pregnane X receptor tightens the blood-brain barrier to methadone through P-glycoprotein up-regulation. Mol Pharmacol 70(4):1212-1219.

COX-1 Effects on Airway Responsiveness and Allergic Inflammation

In an NIEHS-funded study, a team of intramural researchers has demonstrated beneficial effects of constitutive over-expression of cyclooxygenase-1 (COX-1) on murine airway responsiveness to cholinergic stimulation - but not the anticipated beneficial effects on inflammatory and functional responses of the lung to an allergic stimulus.

Researchers used transgenic mice that over-expressed human COX-1 to study the roles of COX and COX-derived prostanoids in the lung under normal and inflammatory conditions. Along with invasive and non-invasive analyses of lung function, the researchers quantified prostanoid levels and inflammatory cells and mediators in brochoalveolar lavage fluid to investigate differences between COX-1 transgenic and wild-type control mice. Experiments with mice that were also genetically deficient in COX-2 enabled the researchers to control for the potentially confounding effects of COX-2 up-regulation.

Beneficial effects of COX-1 overexpression were observed on airway responsiveness in naïve mice, but allergic airway inflammation and associated functional alterations were not improved. The researchers proposed that other genetic and pharmacologic strategies designed to alter the airway level of specific prostanoids or to target downstream signaling events may be even more beneficial in improving basal lung function and might prove clinically useful in alleviating allergic airway inflammation and hyper-responsiveness.

Citation: Card JW, Carey MA, Bradbury JA, Graves JP, Lih FB, Moorman MP, Morgan DL, DeGraff LM, Zhao Y, Foley JF, Zeldin DC. 2006. Cyclooxygenase-1 overexpression decreases basal airway responsiveness but not allergic inflammation. J Immunol 177(7):4785-4793.

Combined Method for Studying Key Enzyme in DNA Replication and Repair

NIEHS-funded researchers from the Laboratory of Structural Biology, in collaboration with UNC chemists, have demonstrated the insights to be gained from combining protein crystal structures of DNA polymerase beta with high level Quantum Mechanics/Molecular Mechanics (QM/MM) calculations to better understand the chemistry at the atomic level for correct insertion by DNA polymerase beta (pol beta). The use of QM/MM calculations allows for quantum level calculations at the active site while still taking into account the entire protein/DNA environment. The crystal structure used for the study contained a non-hydrolyzable incoming nucleotide triphosphate. Thus, the starting structure for theoretical analysis contained all the atoms, including the two metal ions, required for catalysis.

Since the starting geometries are very near those of the transition state, it becomes possible to mimic the actual incorporation event. The marriage of the crystallography and theory thus provides unusual insight into the chemistry behind DNA replication.

Citation: Lin P, Pedersen LC, Batra VK, Beard WA, Wilson SH, Pedersen LG. 2006. Energy analysis of chemistry for correct insertion by DNA polymerase beta. Proc Natl Acad Sci U S A 103(36):13294-13299.

Engineering RNA-Binding Proteins

Two DIR researchers in the NIEHS Laboratory of Structural Biology have demonstrated the potential for using a Puf family protein, Pumilio1, as a scaffold for engineering RNA-binding proteins with a designed sequence specificity. Puf family proteins regulate germ-line stem cell development, a role they perform both in lower organisms and likely in humans.

Researchers created mutations of Homo sapiens Pumilio 1 homology domain (hsPUM1-HD) to alter sequence specificity and performed RNA-binding experiments to determine binding specificity for cognate RNA compared to wild-type RNA. In many, but not all cases, the team was successful in designing mutant proteins with binding affinity as great or greater than wild-type proteins.

The ability to engineer hsPUM1-HD RNA sequence specificity may help scientists to probe processes that are critical for producing correct protein products in the right amounts at the right times and places. The ability to recognize specific nucleotide sequences could ultimately result in development of interventions with potential for controlling and/or correcting splicing defects that can lead to cancer and other disease and inhibiting overproduction of tumor necrosis factor alpha, which can trigger a range of inflammatory disorders.

Citation: Cheong CG, Hall TM. 2006. Engineering RNA sequence specificity of Pumilio repeats. Proc Natl Acad Sci U S A 103(37):13635-13639.

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