Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Veterinary Physiology, Pharmacology, and Toxicology (Veterinary Medical Sciences)

First Advisor

Jay C. Means

Abstract

Investigations were conducted to identify biomarkers of exposure to dinitropyrenes (DNPs), which are mutagenic and carcinogenic residues of incomplete combustion. One possible biomarker, a DNP-protoporphyrin IX adduct, was not found in analytically useful quantities in liver tissue or in bile. Clastogenicity, a biomarker of effect, also was found to be absent in marrow cells following subchronic exposure of rats to DNPs by gavage. Solubility studies, used as a basis for dosing guidelines, revealed that DNPs are relatively insoluble in both aqueous and lipid tissue compartments. Calculated log P values indicated that the reduced metabolites of DNPs probably are more soluble in tissues than are DNPs. Thus, increasing solubility may drive nitroreduction. The dependence of expression of a DNP-related biomarker on nitroreductase activity led to efforts to quantify nitroreductase activity. A nitroreductase assay of hepatic cytosol surprisingly revealed that DNP nitroreduction proceeded according to log (FMN), as well as to enzyme content. Further experiments revealed that flavin-dependent nitroreduction occurred by chemical, rather than enzymatic activity. This reaction should have been unfavorable thermodynamically, and so electrochemical experiments were undertaken to re-measure the electrochemical half-wave reduction potential. Cyclic voltammograms of 1,6-DNP, using a platinum working electrode, vs. Ag/AgCl, yielded a reduction wave with a crest at $-$100 mV. Electrolysis of 1,6-DNP at potentials at least as positive as 0.00 mV vs. Ag/AgCl reference electrode, using platinum or mercury working electrodes, yielded 1-amino-6-nitropyrene and pyrene-1,6-diamine, proving that the wave in cyclic voltammetry was a nitroreduction wave. Thus, DNPs are reduced more easily than previously reported. Flavin-dependent nitroreduction has been studied extensively in certain nitroarene pharmaceuticals, and the nitroreduction mechanism is well understood. Extension of this chemistry to DNPs reveals that the oxidative damage and protein alkylation that accompany the nitroreduction reaction may act as tumor promoters. Thus, direct exposure of tissues to DNP nitroreduction may cause fixation of more mutations than might be expected of exposure to DNP metabolites only. This mechanism may be important to consider relative to tumorigenesis and biomarker studies, and to DNP risk assessment.

ISBN

9780591289251

Pages

145

DOI

10.31390/gradschool_disstheses.6350

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