Date of Award:

5-1994

Document Type:

Dissertation

Degree Name:

Doctor of Philosophy (PhD)

Department:

Animal, Dairy, and Veterinary Sciences

Department name when degree awarded

Toxicology

Committee Chair(s)

Roger A. Coulombe, Jr.

Committee

Roger A. Coulombe, Jr.

Committee

Joseph K. K. Li

Committee

Raghubir P. Sharma

Committee

Frank R. Stermitz

Committee

William A. Brindley

Committee

Ann E. Aust

Abstract

Pyrrolizidine alkaloids are cytotoxic, carcinogenic, and anti-carcinogenic in vivo and in vitro, and they produce many hazardous effects in humans and animals. Pyrrolizidine alkaloids (PAs) also cross-link with DNA and/or protein. However, whether such cross-linking is important to the toxic action of PAs is not known. In addition, the exact mechanism underlying these DNA cross-links or cytotoxicity is also not clear.

In three separate studies, I characterized the nature of PA-induced DNA cross-links and the relationships between PA structures and cross-linking potency. In the first study (Chapter II), I found that cross-linking potency of PA congeners coincided with their abilities to cause cytopathologic effects. Macrocyclic α,β-unsaturated diesters PAs and their pyrrolic metabolites were the most potent inhibitors of colony formation, and inducers of cytopathologic changes and megalocyte formation. The macrocyclic α,β-saturated diester PA and open diesters PAs slightly inhibited colony formation, and slightly changed cell morphology. Retronecine and indicine N-oxide were completely inactive. In the next study (Chapter III), I found that pyrrolic macrocyclic metabolites were more potent DNA cross-linkers than their parent compounds as determined by alkaline elution. The pyrroles of the macrocyclic diester PAs were potent DNA-DNA (inter- and/or intra) cross-linkers in Bst EII-digested λ-phage DNA or pBR322 plasmid DNA but dehydroretronecine and indicine N-oxide were not. I also examined which DNA sequences were more susceptible to PA-induced cross-links by using a series of restriction endonucleases to determine sequence specificity. The most favorable cross-linking site for PAs appeared to be 5'-d(GG) and 5'-d(GA) although other sites, 5'-d(CC) or 5'-d(CG), might be also preferable cross-linking targets. In the next study (Chapter IV), I characterized the nature of DNA-protein interactions induced by PAs, because I found in previous studies that PA-induced cross-links are largely protein associated. In PA or pyrrolic PA exposed cells, cross-linked proteins with molecular weights 40 - 60 kD were detected. Two-dimensional electrophoretic analysis revealed that these proteins were probably acidic in nature. In an in vitro system utilizing pBR322 or Bst EII-digested λ-phage DNA, dehydrosenecionine induced DNA-protein cross-links with BSA, indicating that such interactions might be related to amino acid composition of protein.

These results confirmed that PA-induced DNA cross-links (DNA-DNA, DNA-protein cross-links) are influenced by three structural features: the C1,2 unsaturation of pyrrolizidine ring, α,β-unsaturation, and size of the macrocyclic diester ring. The ability to form cross-links was closely related to the known toxic potencies of these PAs. From this research, I also conclude that DNA crosslinking is the most critical event leading to PA-related diseases and that crosslinking is due to pyrrolic metabolites of PAs, not via a common metabolite as was once thought.

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