Source: UNIVERSITY OF MASS DARTMOUTH submitted to
PESTICIDE IMPACT ON PLANT DNA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
NEW
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0196383
Grant No.
2003-35318-13574
Project No.
MASR-2003-02111
Proposal No.
2003-02111
Multistate No.
(N/A)
Program Code
54.3
Project Start Date
Aug 1, 2003
Project End Date
Jul 31, 2005
Grant Year
2003
Project Director
Boerth, D. W.
Recipient Organization
UNIVERSITY OF MASS DARTMOUTH
285 OLD WESTPORT ROAD
NORTH DARTMOUTH,MA 02747
Performing Department
(N/A)
Non Technical Summary
Use of pesticides in agriculture is important to insure crop viability by thwarting weeds, insects, fungi, and disease. Studies point to genotoxic risk to agricultural plants, as well as humans and animals, from chemical agents used in crop production. Adduct formation, binding of these chemicals with DNA, is strongly implicated. Many pesticides are electrophilic agents which are potentially reactive at various sites in DNA. Reactions of DNA bases with pesticides or their metabolites alter the structure of the nucleic acid and prevent proper replication. This degradation of genetic material leads to inferior development of vine, leaf, roots, or fruit and ultimately to inferior quality or yield of the agricultural product. This study focuses on the degree and nature of the risk to plant DNA from pesticides either through direct interaction with the pesticide molecules or their metabolites or through indirect DNA damage by oxidative stress. Biological experiments are conducted involving pesticide treatment of various crop plants, isolation of plant DNA, and assaying of DNA modifications. These experiments are augmented by molecular modeling at various levels of theory to determine the nature and extent of adduct formation of pesticides with DNA bases. This will permit screening of pesticides to assess risk/benefit to the quantity and quality of crops. The modeling can be applied to the design of new pesticides which pose less threat to crop plants. Therefore, this approach holds promise of enhancing crop production to the benefit of U.S. agriculture.
Animal Health Component
(N/A)
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031121200010%
2031499200010%
2031599200010%
2032410200020%
2035220200050%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
5220 - Pesticides; 1121 - Cranberry; 1599 - Grain crops, general/other; 1499 - Vegetables, general/other; 2410 - Cross-commodity research--multiple crops;

Field Of Science
2000 - Chemistry;
Goals / Objectives
Assay DNA adduct formation as a biomarker for genotoxic and oxidative stress by pesticides and their metabolites in plant systems, assess the extent of formation of adducts in plant systems, identify the nature and sites of interactions between pesticide molecules and DNA bases, deduce structures of intermediates and products in covalent binding with pesticide molecules, develop theoretical modeling methods for screening pesticide potential for DNA-damage and genotoxicity, correlate theoretically derived parameters and experimental data to develop quantitative structure activity relationships (QSAR) for screening of pesticides, design and propose molecular structures for new, modified pesticides which potentially pose reduced risk of DNA damage and genotoxicity.
Project Methods
A two-pronged approach is employed involving both biological and molecular modeling studies to identify the nature, degree, and site(s) of interactions between DNA bases and pesticide molecules. A variety of crop plants will be grown and be treated with a series of pesticide preparations by application to leaves, stem, fruit, and root systems. DNA from these plants will be harvested by standard procedures. The extent of DNA modification by various pesticides will be established by 32P post-labeling and radiochromatography. Adducts formed between DNA bases and pesticide molecules or metabolites will be synthesized by direct incubation of the nucleosides with the pesticide. Isolation by chromatography followed by spectroscopic characterization will yield structures of these DNA adducts and products for comparison with results from chromatography. These compounds will also confirm the nature of DNA binding and determine the actual site of attack. Parallel molecular modeling of pesticide interactions with DNA bases will be carried out by molecular orbital calculations at various levels. This modeling will investigate potential interactions between pesticide molecules and nucleotide bases. Computer graphical maps of electron distributions and electrostatic potential will be constructed to identify the nature and sites of potential interactions with pesticide molecules. Computed properties (orbital energies, charges, etc.) of pesticide molecules and modified analogs will be explored to provide QSAR correlations of interactions with nucleic acids leading to potential genotoxicity. Modeling software for intermolecular interactions utilizing an electrostatic multipole technique, currently under development in our laboratories, will be applied to the study of binding between pesticide molecules and DNA bases.

Progress 10/01/03 to 09/30/04

Outputs
Experimental studies continue to determine risk potential for pesticides in agricultural plants, 1) genotoxicity and 2) oxidative stress to crops from pesticide use. These aspects are being explored by assaying plant DNA adducts as biomarkers for genotoxic and oxidative stress in crop plants. The following crop plants have been treated with a series of pesticides: bush beans, soybeans, cucumber, grapes, pumpkin, and tomatoes. A number of additional species are currently under investigation, including cranberries and wheat. The agents used thus far, chlorothalonil, diazinon, esfenvalerate, and carbaryl, are being expanded to other pesticides. DNA isolated from these plants was subjected to 32P post-labeling and radiochromatography. Adducts were found for all treated plants compared with untreated plants. Direct adducts with the chemical agent or metabolites were found for soybeans with chlorothalonil and diazinon. Indirect adduct formation with deoxyguanosine (dG) was found in other cases. 2-Hexenal(HXL)-dG and 4-hydroxy-2-nonenal (HNE)-dG derivatives were found for chlorothalonil in bush beans, cucumbers, and pumpkins, with lesser amounts in grapes and soybeans. Soybeans also yield both HXL-dG and HNE-dG adducts with esfenvalerate, diazinon, and carbaryl. The presence of these adducts indicates that these plants exhibit oxidative stress from pesticide agents via lipid peroxidation by formation of HXL and HNE from arachidonic acid by cyclooxygenases. The presence of HXL and HNE adducts with DNA serve as good biomarkers for stress to the plant from pesticide use. The presence of DNA adducts from direct reaction with the pesticide of its metabolites or from the products of lipid peroxidation is an indication that DNA base damage has occurred in these treated plants with the potential of mutation in the plant. To further explore the formation of adducts particularly by direct reaction, in vitro investigations are being undertaken. A series of pesticides are reacted with deoxyguanosine, the most reactive of the DNA bases, in phosphate buffered aqueous methanol solutions. The reaction product mixtures are then subjected to high pressure liquid chromatography (HPLC) to separate the adducts from guanosine and pesticide compounds. Evidence has been found for direct adduct formation of guanosine with chlorothalonil. Work is continuing with several other pesticides. Attempts are being made to isolate and characterize these products. In addition, computational modeling of reactive potential between DNA and pesticide molecules has been carried out via quantum calculations using semi-empirical, ab initio, and density functional theory (DFT). Electrostatic potential plots of a variety of pesticides (chlorothalonil, dichlobenil, sethoxydim, simazine, napropamide) have been evaluated and used to inform subsequent calculations of interaction energies between guanosine and pesticide molecules. DFT interaction energies are in the range of 1.0-3.5 kcal/mol. These are comparable to other interaction energies for known DNA alkylating reagents, indicating considerable potential for DNA adduct formation and damage.

Impacts
These studies are significant in advancing the knowledge of the effects of pesticides on plant DNA. They have already demonstrated that crop plants are susceptible to genotoxic and oxidative stress from treatment with certain chemical agents. To our knowledge, this is the first identification of DNA adducts from products of lipid peroxidation in plants. The presence of DNA adducts is very useful biomarker for oxidative stress, as well as genotoxic damage. Quantitation of the DNA adducts, wherever practicable, will provide data on the degree and nature of the risk of pesticides to plant DNA. This will be the subject of ongoing studies to determine which pesticides are safer and less prone to promoting genotoxic and oxidative stress. This study provides an assessment of pesticide risk/benefit by identifying which pesticides present the hazard of DNA damage to the quantity and quality of crops. This information will provide growers with additional data to augment existing programs of Integrated Pest Management. Coupling of the experimental work with computational modeling studies will provide quantitative structure-activity information which will be useful in suggesting putative chemical structures for compounds which pose decreased risk to plant DNA. Synthesis of these compounds in the future could conceivably lead to patentable pesticides after further testing. This work has led to three papers at American Chemical Society National Meetings in the Division of Agrochemicals. A paper in the Journal of Agricultural and Food Chemistry is also forthcoming.

Publications

  • "DNA Adduct Formation as Biomarkers for Oxidative Stress and DNA Damage in Crop Plants Treated with Pesticides," Boerth, D. W., Eder, E., Stanks, J. R., Wanek, P., Wacker, M., Gaulitz, S., Skypeck, D., Pandolfo, D., and Yashin, M. J. Agric. Food Chem., submitted, 2005.
  • "In Vitro Formation Of Guanosine Adducts With Pesticides" Boerth, D. W. ; Melissa Medeiros, M.; Coulombe, D. Accepted, 229th ACS National Meeting, Division of Agrochemicals, Paper No. AGRO-xxx, March 14, 2005.
  • "Assessing Risk Of Genomic Damage In Crops From Pesticides" Boerth, D. W.; Eder, E.; Stanks, J. R.. Abstracts of Papers, 228th ACS National Meeting, Division of Agrochemicals, Paper No. AGRO-050, August 24, 2004.
  • "DNA Adduct Formation From Pesticides", Boerth, D. W.; Stanks, J. R.; Eder, E.. Abstracts of Papers, 227th ACS National Meeting, Division of Agrochemicals, Paper No. AGRO-047, March 29, 2004.