Date Awarded

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Virginia Institute of Marine Science

Advisor

Peter A. Van Veld

Abstract

The mammalian mdr1 P-glycoprotein (Pgp) has been implicated in xenobiotic resistance of drug-resistant cell lines and tumors, and may function in excretion or exclusion of toxic xenobiotics at several sites within the body. Pgp gene family members are expressed in excretory epithelia of several aquatic taxa and may contribute to the survival of pollution-tolerant aquatic animals in contaminated environments. For this reason the expression of Pgp family members was investigated in the livers and liver tumors of creosote-resistant mummichog (Fundulus heteroclitus) inhabiting a creosote-contaminated site (Atlantic Wood) in the Elizabeth River, Virginia. Expression of members of the Pgp gene family was detected by immunochemical methods using monoclonal antibody (mAb) C219. An immunoreactive band in the size range of mammalian P-glycoproteins was observed in immunoblots of liver membrane fractions and detergent extracts of mummichog liver from both Atlantic Wood and a reference site. Immunohistochemical staining of mummichog liver demonstrated this antigen on the canalicular surface of hepatocytes in normal liver similar to expression in mammalian and guppy liver. The levels of Pgp were 2-3 fold higher in immunoblots of nontumorous livers of Atlantic Wood mummichog compared with levels in reference site fish. Similar to results reported in mammalian liver neoplasms, Pgp was overexpressed and had aberrant immunohistochemical localization in the majority of mummichog liver neoplasms. Because mAb C219 was expected to recognize all Pgp gene family members expressed in mummichog, molecular techniques were used to characterize the individual Pgp genes expressed in mummichog liver. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify Pgp gene transcripts. A cDNA fragment (3004 bp) was amplified which was most similar to the recently described mammalian sister gene to P-glycoprotein (spgp). Northern blots showed predominant expression of this gene in mummichog liver. Another cDNA fragment (1713 bp) was amplified from mummichog liver and intestine which was a homolog of the mammalian multidrug-resistance or xenobiotic transporter (mdr1). These results indicate that a xenobiotic transporter may be elevated in the livers and liver tumors of pollution-tolerant mummichog consistent with the proposed role of these proteins in xenobiotic resistance.

DOI

https://dx.doi.org/doi:10.25773/v5-afeh-j139

Rights

© The Author

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