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Genetic Studies
Project Summary

Title: DNA Damage from Chemical Agents and Its Repair (Project IV)
Synopsis: This animal and human study aims to determine the long-term consequences of Chemical Weapon exposure on nervous and non-nervous system tissue, and to establish a possible link between those exposures and DNA damage and repair.
Overall Project Objective: Determine the long-term consequences of genotoxin exposure on nervous system and non-nervous system tissue.
Status/Results to Date: 1) We studied the response of neuronal and glial cells and human skin tissue to HN2. Human and rodent postmitotic neurons proved to be more sensitive to HN2 than glial cells. 2) We examined the acute (24 h) and long-term (7 day) cytotoxic effect of HN2 on human skin tissue (SKIN2(tm)). HN2 is acutely toxic to human skin tissue at high concentrations but extensive tissue damage can be induced by lower concentrations (1/10 or 1/100 of the acute dose) when tissue is exposed for long periods of time. 3) We developed assays to measure the predominant HN2-induced DNA adduct (monofunctional; GN7) and cross-links. We demonstrated that guanine-N7(GN7) levels were 2-fold higher in postmitotic rat cerebellar neurons than dividing SY5Y cells comparably treated. We showed that cross-link levels in HN2-treated human SY5Y neuroblastoma cells increased with increasing concentrations of HN2. Studies are underway to determine if these adducts persist in HN2-treated nervous tissue. 4) We developed an assay for examining the relationship between cytotoxicity and DNA damage. 5) We developed assays for measuring the effect of HN2 on neuronal and human skin tissue DNA-repair levels and activity. 6) Nitrogen mustard (HN2) modulated the level and activity of the nervous tissue DNA-repair protein APE, which specifically repairs the monofunctional HN2 DNA adduct. Apurinic apyrimidinic (APE) levels increased in HN2-treated rodent neuronal cultures (non-dividing cells), while APE levels decreased in human neuronal cultures (dividing cells). APE activity increased in HN2-treated human skin cultures (actively dividing cells). Altered levels and activity of APE may be an indication of HN2-induced DNA damage. 7) Nitrogen mustard (HN2) modulated the level and activity of APE in human skin tissue. A significant increase in immunostaining was demonstrated in human skin probed with an antibody to APE. APE activity was increased in cultured human skin explants treated with HN2. 8) Lymphocytes from a small group of psychologically stressed PGW veterans were examined for oxidative DNA repair (i.e., APE and OGG levels and activity). APE levels and activity were increased in lymphocytes of subjects exhibiting psychological stress with accompanying somatic symptoms. The same lymphocyte samples were analyzed for APE and 8-oxodeoxyguanosine DNA glycosylase (OGG) by a newly developed oligonucleotide assay, but no activity could be found (inefficient amount of sample). Recent studies explored the effect of repetitive stress on brain tissue DNA repair. Coronal and saggital brain tissue sections of stressed and unstressed mice were immunoprobed with an antibody to APE (previously used to determine human lymphocyte APE levels) and a significant decrease in immunostaining was observed in the hippocampus of stressed mice when compared with unstressed mice. These studies suggest that stress (e.g., psychological) may perturb brain tissue DNA repair. 9) Cerebellar granule cell cultures prepared from mice defective in nucleotide excision DNA repair [i.e., heterozygous (+/-) and homozygous (-/-) for the Xpa protein] were treated with HN2 and lactate dehydrogenase (LDH) release was found to be 1.5-3.0x higher in -/- mice than +/- mice. A similar effect on LDH release was observed when neurons from -/- mice were exposed to UV irradiation. These studies demonstrate that nucleotide excision DNA repair plays an important role in protecting neurons from HN2-induced cytotoxicity, possibly by the formation of HN2 DNA cross-links.
Project:VA-6D
Agency:Department Of Veterans' Affairs
Location:VAMC Portland
P.I. Name:Glen Kisby, Ph. D., Peter Spencer, Ph. D.
Research Type:Mechanistic
Research Focus:Chemical Weapons
Focus Category:Genetic Studies
Status:Complete
Study Start Date:October 01,1994
Estimated Completion Date:March 31,2000
Specific Aims: 1) Determine the type and quantity of specific DNA adducts and the capacity to repair these adducts in control and nitrogen mustard (HN2) treated genomic DNA isolated from normal human skin and brain; 2) Determine the type and quantity of specific DNA adducts and the capacity to repair these adducts in genomic DNA isolated from mouse cerebral cortex cultures treated with HN2; 3) Assess the relationship between DNA damage, DNA repair and cell degeneration in rodent neuronal cultures treated with HN2; 4) Compare and contrast DNA damage, DNA repair and cytotoxicity in primary neuronal and primary glial cultures treated with HN2.
Methodology: 1) Determine the relative sensitivity of different nervous tissue cell types to HN2. Nervous tissue cell cultures (e.g., rodent astrocytes, cerebellar granule cells, cortical explants, and SY5Y human neuroblastoma cells) will be treated for various time periods with a range of HN2 concentrations and examined for cytotoxicity using fluorescent dyes for live cells (e.g., the mitochondrial fluorochrome Rhodamine 123) and dead cells (e.g., the nuclear DNA-binding fluorochrome propidium iodide). 2) Develop sensitive methods for the detection of DNA adducts (i.e., monofunctional and cross-links) in HN2-treated nervous tissue and human skin explants. The most sensitive methods for detecting HN2-induced DNA adducts are the 32P-postlabeling/TLC (monofunctional adducts) and ethidium bromide assay (crosslinks). The 32P-postlabeling/TLC and ethidium bromide assay method was developed and used to detect DNA adducts in tissue treated for periods up to 7 days with HN2. 3) Develop methods for determining DNA-repair levels and activity in mustard-treated neuronal (e.g., cortical, cerebellar) and non-neuronal (e.g., astrocytes) cultures and human skin. The predominant DNA adduct produced by sulfur and nitrogen mustards is the alkylated guanine DNA adduct. The alkylated guanine DNA adduct is repaired by the base-excision repair (BER) pathway. The rate-limiting step in BER is the repair of an apurinic site by apurinic/apyrimidinic endonuclease (APE). APE levels and activity in nervous tissue treated with HN2 will be determined by western blotting and a method using damaged plasmid DNA. These studies will determine the relative DNA repair of HN2-induced DNA adducts in different CNS cell types. Findings from these studies will be important for establishing a possible relationship between DNA damage, genotoxin exposure and cytotoxicity in nervous tissue.
Most Recent Publications:

Kisby G, Davis E, Springer N, Spencer P. Determination of nitrogen mustard-induced DNA adducts in nervous tissue by an HPLC method with electrochemical detection. N/A, Submitted 4/99; Under Revision. Article

Kisby G, Springer N, Spencer P. Differential sensitivity of nervous tissue cell types to nitrogen mustard-induced toxicity. N/A, In Preparation. Article

Kisby G, Springer N, Spencer P. In Vitro neurotoxic and DNA-damaging properties of nitrogen mustard (HN2). J Appl Toxicol, 20 Suppl 1:S 35-41, Dec 2000. Abstract

Spencer P, Daniels J, Kisby G. Mustard warfare agents and related substances. Experimental and Clinical Neurotoxicology, PP 837-848, 2000. Article

Kisby G, Springer N, Spencer P. Nitrogen mustard damages Neuronal DNA and alters DNA repair. N/A, In Preparation. Article

Kisby G, Sweatt, Spencer P. Role of DNA repair in protecting mature nervous tissue from DNA damage. J Cell Biochem, 21 A, 348, 1995. Article