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July 2011

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Intramural papers of the month

By Raluca Dumitru and Ian Thomas
July 2011

Diet may protect against mutagens in fried meat

Investigators from NIEHS, along with collaborators from the University of North Carolina at Chapel Hill, the Environmental Protection Agency, and several other institutions, have found that dietary factors can reduce DNA damage caused by heterocylic amines (HCAs), carcinogenic compounds formed in meat cooked at high temperatures.

The researchers determined that eating cruciferous vegetables, such as broccoli and cauliflower, chlorophyll-derived chlorophyllin (CHL) tablets, and yogurt reduced the amount of DNA damage found in colon cells obtained by biopsy, compared to the tissue of other volunteers on a control diet. The study is the first to show that eating these foods can measurably reduce DNA damage in human colon cells.

Sixteen volunteers were divided into two groups of eight. Members of one group were randomly assigned to a diet containing meats cooked at a low (100° C) or high temperatures (250° C). Participants in the other group were randomly assigned to diets containing high-temperature meat alone or in combination with the inhibitors. Within each group, members switched diets after two weeks so that each person could serve as their own control. Blood, urine, and colon biopsies were obtained from participants each week over the four week study.

Urine and fecal samples from the low-temperature meat diet had low levels of mutagenicity, while samples from the high-temperature meat diet displayed significantly increased levels of mutagenicity. Fecal mutagenicity and colon cell DNA damage was significantly decreased in those who added the inhibitors to their diets. 

While additional subjects and longer dietary regimens are recommended for future studies, this work establishes an important link between diet and the reduction of DNA damage in the colon. 

Citation: Shaughnessy DT, Gangarosa LM, Schliebe B, Umbach DM, Xu Z, Macintosh B, Knize MG, Matthews PP, Swank AE, Sandler RS, Demarini DM, Taylor JA.( Exit NIEHS 2011. Inhibition of fried meat-induced colorectal DNA damage and altered systemic genotoxicity in humans by crucifera, chlorophyllin, and yogurt. PLoS One 6(4):e18707. Story (

Formation and repair of double strand breaks in yeast

A recent NIEHS study is the first to demonstrate that in the budding yeast Saccharomyces cerevisiae, single-strand breaks (SSBs) generated by the alkylating agent methyl methanesulfonate (MMS) can lead to double-strand breaks (DSBs) in the DNA of G2/M arrested cells. In addition, the research team identified a novel repair intermediate, called slow-moving DNA (SMD), using pulsed field gel electrophoresis (PFGE). Until the publication of this research, there was no direct evidence of the formation and subsequent repair of MMS-induced DSBs in vivo. This report provides an informational leap in understanding genomic instability.

The investigators treated yeast mutants lacking apn1/2 endonuclease with MMS and detected end-processing of random DSBs using "PFGE-shift," a technique developed in their lab. Since the mutants lacked endonuclease, and were therefore unable to perform base excision repair, the damage led to the creation of apurinic/apyrimidic (AP) sites and subsequently to 3' blocked SSBs. Additional mutations that affected AP sites decreased the number of DSBs. The formation of SMD was independent of resection/recombination processes.

Since approximately 10,000-200,000 SSBs are thought to occur in mammalian cells each day, even a fraction of them turning into DSBs could significantly affect genome stability. These findings provide a greater understanding of the role that DNA damage may play in cancer and cell death.

Citation: Ma W, Westmoreland JW, Gordenin DA, Resnick MA. ( Exit NIEHS 2011. Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease. PLoS One 7(4):e1002059.

Crystal structure-based mutagenesis of EndA nuclease

Investigators at NIEHS, the University of North Carolina at Chapel Hill and Justus-Liebig-University in Giessen, Germany have elucidated the X-ray crystal structure of EndA nuclease at 1.75 Å, and, in doing so, have determined the amino acids involved in its substrate binding and nuclease activity. The study is the first to use site-directed mutagenesis based on structural data to negate DNA binding by EndA nuclease, a member of the ββα-metal finger superfamily. EndA nuclease contributes to the virulence of Streptococcus pneumonia, which can cause serious respiratory illnesses. The end goal of this project was to lay the foundation for advances in the production of antimicrobial therapeutics to treat infection.

After determining the crystal structure, the researchers focused on the EndA catalytic site, which exhibited an Asp-Arg-Gly-His motif-containing ββα-metal finger core. Based on their proximity to the nuclease active site, several residues were substituted with alanine to uncover their role in catalysis and binding. These studies identified His154, Gln186, Asn191, Gln192, and Glu205 as being necessary for catalysis, with Asn191 and Glu205 being the most crucial. Additionally, the team found that Arg127/Lys128 and Arg209/Lys210 were necessary for substrate binding.

This study provides an important glimpse into the structure of S. pneumoniae EndA nuclease, providing critical information about how it binds the host's DNA. Since EndA nuclease destroys one of the body's defenses against the bacterium, known as neutrophil extracellular traps (NETs), specific inhibitors designed against EndA could help in the treatment of S. pneumoniae infection and have significant implications for human health.

Citation: Moon AF, Midon M, Meiss G, Pingoud A, London RE, Pedersen LC. ( Exit NIEHS 2011. Structural insights into catalytic and substrate binding mechanisms of the strategic EndA nuclease from Streptococcus pneumoniae. Nucleic Acids Res 39(7):2943-2953.

A novel protective role for beta2 adrenergic receptor in Parkinson's disease

A new research study stemming from a collaboration between researchers from NIEHS and the University of North Carolina at Chapel Hill uncovered a novel protective function for the beta2 adrenergic receptor (beta2AR) in Parkinson's disease. Team members gave mice salmeterol, a beta2AR agonist commercially known as Advair, and found that it protected the rodent brains against inflammation. This effort is the first to show that brain inflammation associated with Parkinson's disease can be effectively decreased using beta2AR agonists.

Chronic neurodegenerative inflammation has been long believed to play a major role in the progression of Parkinson's disease and studies have shown that microglia, the macrophages of the brain, are important mediators of inflammation.

To mimic the symptoms seen in Parkinson's disease, the investigators treated mouse models and neuron-cell glia cultures with the bacterial endotoxin lipopolysaccharide and the neurotoxin MPTP, to cause inflammation associated with Parkinson's. The scientists treated the mice with salmeterol, and saw that even at very low doses (1-10 milligram per kilogram of weight), salmeterol protected the dopaminergic neurons against inflammation. Most importantly, the study further demonstrated that salmeterol reduced the release of pro-inflammatory factors from the microglia.

Although salmeterol and formoterol (Symbicort) are currently being used to treat other inflammatory conditions, such as asthma and chronic obstructive pulmonary disease, they may also be utilized to treat neurodegenerative diseases.

Citation: Qian L, Wu HM, Chen SH, Zhang D, Ali SF, Peterson L, Wilson B, Lu RB, Hong JS, Flood PM. ( Exit NIEHS 2011. β2-adrenergic receptor activation prevents rodent dopaminergic neurotoxicity by inhibiting microglia via a novel signaling pathway. J Immunol 186(7):4443-4454.

(Raluca Dumitru, M.D., Ph.D., is an Intramural Research Training Award fellow in the Cell Biology Group of the Laboratory of Molecular Carcinogenesis. Ian Thomas is a writer/editor in the NIEHS Office of Communications and Public Liaison.)

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