The group continued to implement and validate the technique called immuno-spin trapping, which combines the specific free radical reactivity of the DMPO (5,5-dimethyl-1-pyrroline N-oxide) spin trap with nitrone-antibody sensitivity. Anti-DMPO has proven itself to be a highly specific antibody with the no-DMPO control easily identifying any non specificity issues. This type of control is uncommon among antibody work, but is of great utility. Unlike electron spin resonance (ESR) detection of radical intermediates, anti-DMPO immuno-spin trapping is not dependent on transient free radical intermediates and is about 10,000 times more sensitive than ESR and 100 times more sensitive than MS. In years we have continued using the immuno-spin trapping approach to investigate the formation of protein-centered free radicals in vitro, in cells, and in vivo. The immunoassay has many advantages compared to ESR spin-trapping: 1) immuno-spin trapping requires much less material (micrograms of protein rather than milligrams); 2) the stability of the final oxidation product, the DMPO-nitrone adduct (a stable non-radical species), is much greater than that of the paramagnetic DMPO-radical adducts (t1/2 = sec-min) required for ESR, resulting in greatly enhanced sensitivity, and 3) immuno-spin trapping can be performed using standard ELISA, Western blotting techniques, and immuno-histological techniques, making immuno-spin trapping far more accessible and versatile than ESR spin trapping. This advance greatly expanded the utility of the spin-trapping technique, freeing it from the quantum mechanical complexity of ESR spectroscopy. We found that expression of fluorescent proteins such as enhanced green fluorescent protein (eGFP) induces oxidative stress in cells. The increased formation of reactive oxygen species (ROS) such as superoxide (O2) and hydrogen peroxide (H2O2) can explain the cytotoxicity and tissue abnormalities reported previously in mammalian and bacterial cells and animals overexpressing fluorescent proteins. Both O2 and H2O2 induce redox signaling mechanisms, leading to altered gene expression of cell regulatory proteins involved in cell proliferation, cell differentiation, and cell death. Thus, these findings could have a major influence on the interpretation of results obtained from numerous studies that routinely use fluorescent protein tags. In the Maneb and the herbicide paraquat induced Parkinsons disease model, we found that the cytochrome c plays crucial roles in -synuclein radical formation and oligomerization. Extensive evidence were provided that -synuclein affects several biological pathways, which ultimately contribute to neuronal death in the Maneb and the herbicide paraquat induced mouse model of Parkinsons disease. The presence or absence of -synuclein (in wild-type or -synuclein knockout mice, respectively) determined all the difference in response to Maneb and paraquat co-exposure. These results suggest that experimental approaches which rely on scavenging -synuclein radical or decreasing -synuclein levels, can possibly lead to development of potential therapeutics for Parkinsons disease.

Project Start
Project End
Budget Start
Budget End
Support Year
24
Fiscal Year
2018
Total Cost
Indirect Cost
Name
U.S. National Inst of Environ Hlth Scis
Department
Type
DUNS #
City
State
Country
Zip Code
van 't Erve, Thomas J; Lih, Fred B; Kadiiska, Maria B et al. (2018) Elevated plasma 8-iso-prostaglandin F2? levels in human smokers originate primarily from enzymatic instead of non-enzymatic lipid peroxidation. Free Radic Biol Med 115:105-112
Kumar, Ashutosh; Triquigneaux, Mathilde; Madenspacher, Jennifer et al. (2018) Sulfite-induced protein radical formation in LPS aerosol-challenged mice: Implications for sulfite sensitivity in human lung disease. Redox Biol 15:327-334
Ganini, Douglas; Santos, Janine H; Bonini, Marcelo G et al. (2018) Switch of Mitochondrial Superoxide Dismutase into a Prooxidant Peroxidase in Manganese-Deficient Cells and Mice. Cell Chem Biol 25:413-425.e6
Muñoz, M D; Della Vedova, M C; Bushel, P R et al. (2018) The nitrone spin trap 5,5-dimethyl-1-pyrroline N-oxide dampens lipopolysaccharide-induced transcriptomic changes in macrophages. Inflamm Res 67:515-530
Sinha, Birandra K; van 't Erve, Thomas J; Kumar, Ashutosh et al. (2017) Synergistic enhancement of topotecan-induced cell death by ascorbic acid in human breast MCF-7 tumor cells. Free Radic Biol Med 113:406-412
Sinha, Birandra K; Kumar, Ashutosh; Mason, Ronald P (2017) Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells. Biochem Biophys Rep 10:252-259
Ganini, Douglas; Leinisch, Fabian; Kumar, Ashutosh et al. (2017) Fluorescent proteins such as eGFP lead to catalytic oxidative stress in cells. Redox Biol 12:462-468
van 't Erve, Thomas J; Kadiiska, Maria B; London, Stephanie J et al. (2017) Classifying oxidative stress by F2-isoprostane levels across human diseases: A meta-analysis. Redox Biol 12:582-599
Kumar, Ashutosh; Leinisch, Fabian; Kadiiska, Maria B et al. (2016) Formation and Implications of Alpha-Synuclein Radical in Maneb- and Paraquat-Induced Models of Parkinson's Disease. Mol Neurobiol 53:2983-2994
Kumar, Ashutosh; Ehrenshaft, Marilyn; Tokar, Erik J et al. (2016) Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells. Biochim Biophys Acta 1860:1519-27

Showing the most recent 10 out of 87 publications