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Intramural papers of the month

By Jeffrey Stumpf and Negin Martin
October 2010

Estrogen increases chemical uptake by brain

A collaboration between scientists from NIEHS and the University of Minnesota Duluth has led to the discovery of a new mechanism by which estrogen regulates blood-brain barrier permeability in the rodent brain. Based on their findings, co-administration of estrogen offers a possible new strategy for delivering chemotherapeutics to cancer cells in the brain. 

The blood-brain barrier is a physiological network that protects the brain from chemicals that circulate in blood, but this system also prevents therapeutic agents from reaching and interacting with tumor cells in the brain. The investigators knew that the breast cancer resistant protein (BCRP) present in the capillaries acted as an efflux pump, limiting the brain's uptake of drugs. When team members examined brain capillaries from rodents treated with estrogen, they found that BCRP had been internalized and degraded. They speculated that loss of BCRP should alter the blood-brain barrier to increase brain penetration of certain therapeutic drugs.

This work showed that estrogen signaling at the blood-brain barrier is accomplished through estrogen receptor beta (ERbeta) and activation of the phosphatase and tensin homolog (PTEN) pathway. The signal is transmitted within minutes and does not include the canonical transcriptional activation by the nuclear receptor ERbeta. This research could potentially allow physicians to improve the delivery of chemotherapeutics to the central nervous system, thereby improving the treatment of brain tumors. 

Citation: Hartz AM, Madole EK, Miller DS, Bauer B. ( Exit NIEHS 2010. Estrogen receptor beta signaling through phosphatase and tensin homolog/phosphoinositide 3-kinase/Akt/glycogen synthase kinase 3 down-regulates blood-brain barrier breast cancer resistance protein. J Pharmacol Exp Ther 334(2):467-476.

Base damage of single-stranded DNA intermediate causes localized hyper-mutability in budding yeast

A study from the Chromosome Stability Group at NIEHS demonstrates that the generation of several kilobase regions of single strand DNA (ssDNA) dramatically increases the likelihood of mutations, a phenomenon called localized hyper-mutability (LHM). The paper reports that exposure to the alkylating agent methyl methanesulfonate causes a 20,000-fold increase in mutations near an induced double-strand break (DSB) in budding yeast.

LHM is proposed to occur when ssDNA is exposed to DNA damaging agents resulting in mutagenic replication of irreparably damaged bases. The ssDNA regions are transient intermediates during DSB repair. Therefore, during DSB repair, the adjacent DNA becomes vulnerable to increased mutagenesis. The mutations mostly result from copying of ssDNA-specific methylated lesions of cytosine by DNA pol zeta, a polymerase that often performs error-prone translesion DNA synthesis.

Mutations are an important source of improved evolutionary fitness, as well as precursors for cancer and genetic disease. The increase in mutations needed to drive these processes are thought to be too large to maintain viability, if the mutagenesis is randomly distributed across the genome. The clustered nature of LHM can provide drastic changes to small regions of DNA including multiple changes in single genes, while maintaining overall genome stability.

Citation: Yang Y, Gordenin DA, Resnick MA. ( Exit NIEHS 2010. A single-strand specific lesion drives MMS-induced hyper-mutability at a double-strand break in yeast. DNA Repair (Amst) 9(8):914-921.

Genetic biomarkers for early diagnosis of lung cancer

NIEHS researchers have developed a method for identifying single nucleotide polymorphisms (SNPs) in the human genome that may contribute to lung cancer by affecting gene expression. 

Chronic exposure to cigarette smoke severely injures airway cells yet only 10 to 15 percent of smokers develop lung cancer, suggesting a role for genetic variation in susceptibility. In collaboration with Avrum Spira, M.D., of Boston University School of Medicine, NIEHS researchers obtained samples from patients being screened for lung cancer. Global gene expression was assessed in normal airway cells obtained by bronchoscopy from smokers who developed lung cancer, smokers without lung cancer, and never smokers. Using bioinformatics analysis, differences in NRF2-mediated antioxidant pathway gene expression were observed among these groups and a key role was identified for MAFG, a binding partner of NRF2. 

Hypothesizing that SNPs could contribute to gene expression variability, SNPs in NRF2 binding sites were compared with gene expression. Several SNPs were associated both with gene expression and with cancer status, suggesting that these SNPs could influence expression, and that expression might influence the development of cancer. 

While limited in scope, this work demonstrates a general approach that integrates bioinformatics analysis of pathways and transcription factor binding sites with genotype, gene expression and disease status, to identify polymorphisms affecting individual differences in gene expression and disease risk.

Citation: Wang X, Chorley BN, Pittman GS, Kleeberger SR, Brothers J 2nd, Liu G, et al. ( Exit NIEHS 2010. Genetic variation and antioxidant response gene expression in the bronchial airway epithelium of smokers at risk for lung cancer. PLoS One 5(8):e11934.

Eosinophil peroxidase reacts with sulfite to form protein radicals

NIEHS scientists reported in the Journal of Biological Chemistry that sulfites, hydrated sulfur dioxide, cause oxidative damage to proteins. The study showed that proteins are damaged upon exposure to reactive molecules called free radicals that form during the oxidation of sulfites by eosinophil peroxidase (EPO).

Sulfites are converted to the sulfur trioxide radical by EPO and are further oxidized to form peroxymonosulfate and sulfate anion radicals. Using cell culture cells that produced EPO and detection of trapped radicals in a stable compound by using the spin trap called DMPO, the researchers demonstrated the presence of these reactive radical species and the oxidation of human serum albumin and EPO itself, implying that many other proteins may potentially suffer oxidative damage.

In addition to air pollution, sulfites are found in preservatives, bleaching agents, and various medications.  In the United States, approximately 500,000 individuals are sensitive to sulfite-containing products. Although the mechanism is unknown, toxic levels of sulfites cause allergic reactions similar to those experienced during asthma attacks. Because EPO is secreted by white blood cells, this study raises the possibility that protein damage by free radicals contributes to tissue injury in inflammation and other immune responses.

Citation: Ranguelova K, Chatterjee S, Ehrenshaft M, Ramirez DC, Summers FA, Kadiiska MB, et al. ( Exit NIEHS 2010. Protein radical formation resulting from eosinophil peroxidase-catalyzed oxidation of sulfite. J Biol Chem 285(31):24195-24205.

(Jeffrey Stumpf, Ph.D., is a postdoctoral fellow in the NIEHS Laboratory of Molecular Genetics Mitochondrial DNA Replication Group. Negin Martin, Ph.D., is a biologist in the NIEHS Laboratory of Neurobiology's Viral Vector Core.)

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