Bioassay for toxic substances activated by metabolic enzyme system
AssigneeWisconsin Alumni Research Foundation
InventorsHarry W. Read, Karl Gustavson, George A. Blondin
Life of this patent
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Abstract
A method for bioassaying for metabolic activation of toxicants from a xenobiotic compound by a metabolic enzyme system includes incubating the xenobiotic compound with a metabolic enzyme system known to produce toxicants during normal metabolic degradation processes and with a mitochondrial membrane preparation competent for enzymatic electron transfer. The production of a toxicant has a detrimental effect upon the electron transfer activity of the mitochondrial membrane preparation which can readily be assayed by observing changes in concentration of a selected redox indicator.
Claims43
A method for detecting a metabolically-activated toxicant of a xenobiotic compound in an environmental sample to be tested, the method comprising the steps of:
- (a) incubating a mixture comprising a mitochondrial membrane suspension comprising competent electron transport enzyme complexes thereon, the sample to be tested, a metabolic enzyme system competent to transform the xenobiotic compound into at least one metabolically-activated toxicant, and a first redox indicator required for activity of the metabolic enzyme system under conditions of concentration, time, and temperature adequate to accomplish the transformation;
- (b) adding an assay medium and a second redox indicator which can be converted by the mitochondrial electron transport enzyme complexes to another compound while maintaining the first redox indicator in a reduced state;
- and
- (c) assaying for conversion of the second redox indicator, wherein a rate of conversion different from that of a control sample lacking the metabolic enzyme system indicates the existence of a metabolically-activated toxicant of the xenobiotic compound.
A method for detecting a metabolically-activated toxicant of a xenobiotic compound in an environmental sample to be tested, the method comprising the steps of:
- (a) incubating a mixture comprising submitochondrial particles comprising competent electron transport enzyme complexes I and II, coenzyme Q, and ATPase, the sample to be tested, NADPH-dependent cytochrome P-450 detoxifying enzymes, and a regenerating source of NADPH, under conditions of concentration, time, and temperature adequate to form at least one metabolically-activated toxicant from the xenobiotic compound;
- (b) adding to the incubated mixture a buffered assay medium comprising NAD.sup.+, an inhibitor of mitochondrial electron transfer by mitochondrial enzyme complex III, a source of electron flow into enzyme complex II, and an inhibitor of mitochondrial transhydrogenase-driven reduction of NAD.sup.+ while maintaining NADPH in a reduced state;
- (c) determining a baseline concentration of NADH;
- providing ATP to direct an energy-linked transfer of electrons from the source of electron flow to the NAD.sup.+ to form NADH;
- and
- (d) measuring a change in concentration of the NADH over time, wherein a rate of change slower than that of a control sample lacking the metabolic enzyme system indicates the existence of a metabolically-activated toxicant of the xenobiotic compound.
A method for detecting a metabolically-activated toxicant of a xenobiotic compound in an environmental sample to be tested, the method comprising the steps of:
- (a) incubating a mixture comprising submitochondrial particles comprising competent electron transport enzyme complexes I, III and IV, coenzyme Q, and cytochrome c, the sample to be tested, NADPH-dependent cytochrome P-450 detoxifying enzymes, and a regenerating source of NADPH, under conditions of concentration, time, and temperature adequate to form at least one metabolically-activated toxicant from the xenobiotic compound;
- adding to the incubated mixture a buffered assay medium;
- adding NADH;
- determining a baseline concentration of the NADH;
- and
- measuring a decrease in the concentration of the NADH over time, wherein a rate of decrease slower than that of a control sample lacking the metabolic enzyme system indicates the existence of a metabolically-activated toxicant of the xenobiotic compound.
A kit for use in spectrophotometrically assaying for the presence in an environmental sample of a xenobiotic compound capable being metabolically activated into at least one toxicant, the kit comprising:
- a mitochondrial membrane suspension comprising competent electron transport enzyme complexes, a metabolic enzyme system competent to transform the xenobiotic compound into at least one metabolically-activated toxicant, and a first redox indicator required for activity of the metabolic enzyme system under conditions of concentration, time, and temperature adequate to accomplish the transformation;
- a second redox indicator;
- an inhibitor of mitochondrial electron transfer by mitochondrial enzyme complex III;
- a source of electron flow into enzyme complex II;
- and
- an inhibitor of mitochondrial transhydrogenase-driven reduction of the second redox indicator.
A kit for use in spectrophotometrically assaying for the presence in an environmental sample of a xenobiotic compound capable being metabolically activated into at least one toxicant, the kit comprising:
- a mitochondrial membrane suspension comprising competent electron transport enzyme complexes, a metabolic enzyme system competent to transform the xenobiotic compound into at least one metabolically-activated toxicant, and a first redox indicator required for activity of the metabolic enzyme system under conditions of concentration, time, and temperature adequate to accomplish the transformation;
- and
- a second redox indicator.
Dependencies are inferred from the claim text.