Author

Gail L. Drew

Date of Award:

1997

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Animal, Dairy, and Veterinary Sciences

Advisor/Chair:

Roger A. Coulombe Jr.

Abstract

Pyrrolizidine alkaloids (PAs) are natural plant compounds found in hundreds of plant species worldwide and are reported to have cytotoxic, carcinogenic, antimitotic, and gentotoxic activity. PAs are metabolized by the cytochrome P450 (CYP) sytem to the pyrrole or the N-oxide form. They pyrroles are bifunctional electrophillic alkylators that bind cellular nucleophiles such as DNA and proteins and disrupt normal cell processes, including DNA replication and gene transcription, and can cause megalocytosis. The pyrroles dehyrosenecionine (DHSN) and dehydromoncrotaline (DHMO) are among the most potent PA cross-linkers and inducers of megalocytosis. DHSN and DHMO-induced cross-links in cultured normal (MDBK) and neoplastic (MCF7) cells were nalyzed by SDS-PAGE and Western blot and both were found to contain the protein actin. Actin is crucial to DNA replication and is known to be involved in cross-links induced by cis-dichlorodiammine platinum II (cistplatin), a well known cross-linking drug used for the treatment of cancer. Actin cross-linking may explain the antimitotic, megalocytotic, and anticarcinogenic effects of PAs.

Since protein cross-linking is an important mode of action for PAs, we were interested in what characteristics of the protein might make it a good nucleophilic target. Thus, further research was undertaken based on the hypothesis that cysteine residues, and specifically free sulfhydryl groups, are attractive targets for the bifunctional electrophilic alkylators DHSN and DHMO. Nucleophiles were selected for their abundance in the cell, their cysteine content, and their relationship to the documented side effects of PAs. Actin, glutathione (GSH), metallothionein, topoisomerase II, and cysteine were all found to cross-link with DHSN and DHMO in vitro while methionine, with no free sulfhydryl groups, did not cross-link.

Our results support the hypothesis that cysteine residues are a key characteristic of proteins that are cross-linked by PAs. The cross-links could have negative effects to the cell as in the case of binding actin or topoisomerase II to alter normal DNA processes and replication, or beneficial effects such as binding to electrophillic scavengers like GSH or metallothionine as a detoxifying mechanism. the nucleophiles we tested in vitro and found to form cross-links with DHSN and DHMO may help to explain the antimitotic carcinogenic, and anticarcinogenic effects of PAs.

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