Intramural Papers of the Month
By Brant Hamel, Sonika Patial, Jeffrey Stumpf, and Ian Thomas
- Researchers find novel binding target for rotenone
- Novel role for a DNA polymerase in repairing damaged bases in C. elegans
- (Bi)sulfite oxidation prompts neutrophils to produce free radicals
- Synthetic estrogen induces feminization in male mice via ERalpha
Researchers find novel binding target for rotenone
Previous epidemiology studies have shown that chronic exposure to rotenone, a common pesticide, can reproduce Parkinsonism in rodents and increase the risk of Parkinson’s disease in humans. In a new study from the NIEHS Laboratory of Toxicology and Pharmacology, scientists discovered a novel mechanism that explains how this process works.
The investigators transfected COS7 cells with cytoplasmic subunits of the human phagocyte NADPH oxidase (PHOX), p47(phox) and p67(phox), and the membrane subunits, gp91(phox), the catalytic subunit of PHOX, and p22(phox). Their data suggests that the binding of rotenone to gp91(phox) causes a conformational change that allows p67(phox) to bind to the rotenone/gp91(phox) complex. This binding creates a functional oxidase that generates superoxide. Also, the scientists observed that, in response to rotenone, superoxide production in p47(phox)-deficient macrophages still occurs, which means p47(phox) is not required for this mechanism.
This study clarifies that PHOX is a novel target of rotenone and demonstrates a distinct mechanism by which the pesticide rotenone triggers inflammation-mediated excessive production of free radicals. Oxidative stress-related damage to cellular proteins and nucleic acids may be an important mechanism of pesticide-induced degeneration of nerve tissue.
Citation: Zhou H, Zhang F, Chen SH, Zhang D, Wilson B, Hong JS, Gao HM. (http://www.ncbi.nlm.nih.gov/pubmed/22094225) 2012. Rotenone activates phagocyte NADPH oxidase by binding to its membrane subunit gp91phox. Free Radic Biol Med 52(2):303-313.
Novel role for a DNA polymerase in repairing damaged bases in C. elegans
With genomes being constantly bombarded with DNA damaging agents, organisms require proteins that protect the nucleotide alterations that disrupt cellular function. NIEHS researchers identified a wrinkle in a canonical DNA repair pathway that, in many organisms, requires the specialized DNA polymerase pol beta. The study shows the involvement of another specialized DNA polymerase, pol theta, in a critical step in base excision repair.
Base excision repair is a major pathway to clear DNA damage and to avoid cancer-causing lesions. After recognition of the damage, the combined actions of a damage-specific glycosylase and AP endonuclease excise the damaged base and prepare the DNA for a polymerase to replicate the small gap, resulting in a repaired DNA template.
The researchers used the roundworm C. elegans to study base excision repair. C. elegans is used for developmental biology because the fate of every cell is known, making it an important model organism to understand basic life processes. This study shows that base excision repair in C. elegans requires replication by pol theta and that this model organism does not have a gene for pol beta. Although the overall function of pol theta is not well understood, these results suggest pol theta as a viable alternative to pol beta in base excision repair.
Citation: Asagoshi K, Lehmann W, Braithwaite EK, Santana-Santos L, Prasad R, Freedman JH, Van Houten B, Wilson SH. (http://www.ncbi.nlm.nih.gov/pubmed/21917855) 2012. Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase beta. Nucleic Acids Res 40(2):670-681.
(Bi)sulfite oxidation prompts neutrophils to produce free radicals
New research from scientists at NIEHS have determined that the toxicity of (bi)sulfite in patients suffering from asthma and other pulmonary disorders may result from its ability to induce the production of highly reactive free radicals, such as the sulfate and sulfite anion radical in neutrophils. These free radicals can then oxidize proteins inside of the cell and cause cellular damage.
(Bi)sulfite can be formed in the lung by the hydration of sulfur dioxide, a major air pollutant released by the combustion of fossil fuels. It is also used as a preservative and anti-browning agent in many foods. Using electron spin resonance (ESR) spectroscopy, NIEHS researchers demonstrated that the ability of neutrophils to create these compounds was dependent on the myeloperoxidase (MPO) enzyme. MPO is a very abundant protein — almost 5 percent of cell weight. It is secreted from active neutrophils and is known to catalyze the formation of cytotoxic oxidants involved in inflammatory disorders.
Using specific inhibitors of MPO, researchers could not detect radical formation, thus implicating MPO as necessary for the generation of (bi)sulfite-derived free radicals. Researchers hope that the elucidation of this mechanism will lead to future studies to examine the deleterious effects of (bi)sulfite in respiratory disorders.
Citation: Ranguelova K, Rice AB, Khajo A, Triquigneaux M, Garantziotis S, Magliozzo RS, Mason RP. (http://www.ncbi.nlm.nih.gov/pubmed/22326772) 2012. Formation of reactive sulfite-derived free radicals by the activation of human neutrophils: An ESR study. Free Radic Biol Med; doi:10.1016/j.freeradbiomed.2012.01.016 [Online 2 February 2012].
Synthetic estrogen induces feminization in male mice via ERalpha
In a new study, NIEHS scientists found that estrogen receptor alpha (ERalpha) is responsible for mediating the toxic effects of diethylstilbestrol (DES), a potent synthetic estrogen, on the seminal vesicles of male mice inducing feminization. The harmful effects of DES exposure on the development of the male rodent reproductive tract were well known. The results of this study, however, show that the developing male reproductive tract expresses estrogen receptors and is highly sensitive to the toxicological effects of exogenous estrogens through ERalpha.
The authors injected DES at postnatal days 1-5 in wild-type (WT), ERalpha or ERbeta knockout male mice. DES exposure led to a dramatic reduction in the weight of the seminal vesicles and induced the expression of lactoferrin, an estrogen-inducible uterine secretory protein. It also decreased the expression of seminal vesicle secretory protein IV in WT and ERbeta knockout mice. However, these effects were not seen in ERalpha knockout mice, suggesting that ERalpha mediates DES-induced feminization in male mice.
Although the risk of DES exposure in humans during development is relatively low, the results imply that genistein and bisphenol A, two other environmental and industrial estrogenic compounds to which humans are widely exposed, could have similar effects.
Citation: Walker VR, Jefferson WN, Couse JF, Korach KS. (http://www.ncbi.nlm.nih.gov/pubmed/22275727) 2012. Estrogen receptor alpha (ERα) mediates diethylstilbestrol (DES)-induced feminization of the seminal vesicle in male mice. Environ Health Perspect; doi:10.1289/ehp.1103678 [Online 24 January 2012].
(Brant Hamel Ph.D., is an Intramural Research Training Award (IRTA) fellow in the NIEHS Laboratory of Signal Transduction. Sonika Patial, D.V.M., Ph.D., is a visiting fellow in the NIEHS Laboratory of Signal Transduction. Jeffrey Stumpf, Ph.D., is an IRTA fellow in the NIEHS Laboratory of Molecular Genetics. Ian Thomas is a public affairs specialist with the NIEHS Office of Communications and Public Liaison and a regular contributor to the Environmental Factor.)