Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Gary W. Winston

Abstract

The nitroreduction of 4NQO and five structural analogs were studied with respect to radical production and mutagenicity. In the umu assay, 4NQO was the most mutagenic analog. Addition of microsomal and cytosolic protein from induced and uninduced rat liver samples decreased 4NQO mutagenicity in the standard strain (Salmonella typhimurium TA 1535/psk1002) 4NQO mutagenicity was also found to be decreased in the nitroreductase-deficient strain (Salmonella typhimurium TA1535NR/psk1002). In several strains of the Protox$\rm\sp{TM}$ assay 4NQO yielded the highest induction rate as compared to other compounds tested. In the presence of ascorbate 4NQO cytotoxicity, but not mutagenicity, was substantially enhanced. Oxygen consumption catalyzed during NAD(P)H-dependent microsomal electron transport was enhanced in the presence of 4NQO and 4NPO; this rate of enhancement was similar for both of these nitroarenes. Addition of various flavoprotein inhibitors implicated involvement of a flavoprotein in nitroarene-enhanced oxygen consumption. Replacement of microsomal protein with purified flavin-containing enzymes confirmed that one-electron reduction of 4NQO contributed to enhancement of oxygen consumption. The presence of a redox cycle between O$\sb2$ and various nitroarenes was indicated by direct observation of a nitroanion radical signal in anaerobic ESR studies, spin trappable superoxide anion radical adducts in aerobic ESR studies and the extent of oxygen consumption which was stoichiometricly greater with respect to the concentration of nitroarene present in the assay. ESR confirmed production of a nitrogen-centered radical from 4NQO reduction which was assigned, based on hyperfine splitting constants, as a hydronitroxyl radical from 4NOQO and 4HAQO. NAD(P)H-dependent nitroreduction was catalyzed by microsomes and cytosol from several different animal species. Aroclor 1254 was found to induce the nitroreductase activity of rat liver microsomes over uninduced microsomes. Various purified flavoenzymes catalyzed reduction of 4NPO to nitro anion radical, which was detected under anaerobic conditions, in concomitance with superoxide production. Cytosols prepared from normal, nitroreductase-deficient and nitroreductase-enriched (Salmonella typhimurium TA 1535/psk1002/pNM11) bacterial strains from the umu assay catalyzed NAD(P)H-dependent univalent reduction of 4NPO. The nitroreductase-deficient strain had a greater capacity for this function than the other two strains.

ISBN

9780591591781

Pages

199

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