Studies on the metabolism of chlorotrianisene to a reactive intermediate and subsequent covalent binding to microsomal proteins
The studies on chlorotrianisene were conducted to determine whether metabolism of chlorotrianisene occurs via the cytochrome P450 monooxygenase system and whether a reactive intermediate is being formed that is capable of binding covalently to microsomal proteins. ({sup 3}H)-chlorotrianisene was incubated with liver microsomes supplemented with NADPH. At the termination of the incubation, the protein was trapped on a glass filter and the unbound chlorotrianisene was removed by extensive washing of the protein with organic solvent. A dramatic stimulation of covalent binding was demonstrated in microsomes from rats treated with methylcholanthrene (60 fold increase) versus control or phenobarbital treatment. Verification of covalent binding was achieved by localization of radiolabeled bands following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the macromolecules in the incubation mixture. Further analysis of the radiolabeled macromolecules separated on SDS-PAGE revealed that these macromolecules were degraded by protease degradation indicating that the macromolecules were proteins. Further investigations were done to determine the cause of the dramatic stimulation of covalent binding detected in microsomes from methylcholanthrene treated rats versus control or phenobarbital treated rats. Further evidence for the participation of P-450c was obtained with a reconstituted cytochrome P-450 system. Incubations of chlorotrianisene with reconstituted P-450c and NADPH-cytochrome P-450 reductase exhibited covalent binding characteristics comparable to those seen in microsomal incubations. Investigations into the nature of the binding site and the reactive intermediate are currently being conducted. By analyzing the BSA adduct, the author intends to isolate the specific amino acid binding site(s).
- Research Organization:
- BKI Associates, Inc., McLean, VA (USA)
- OSTI ID:
- 5735480
- Resource Relation:
- Other Information: Thesis (Ph. D.)
- Country of Publication:
- United States
- Language:
- English
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63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
3-METHYLCHOLANTHRENE
BIOLOGICAL EFFECTS
ESTROGENS
METABOLISM
BIOCHEMICAL REACTION KINETICS
ELECTROPHORESIS
LIVER
MICROSOMES
NADP
OXIDOREDUCTASES
PHENOBARBITAL
PROTEINS
RATS
RECEPTORS
TRACER TECHNIQUES
TRITIUM COMPOUNDS
ANESTHETICS
ANIMALS
ANTICONVULSANTS
AROMATICS
AZINES
BARBITURATES
BODY
CELL CONSTITUENTS
CENTRAL NERVOUS SYSTEM DEPRESSANTS
COENZYMES
DIGESTIVE SYSTEM
DRUGS
ENZYMES
GLANDS
HETEROCYCLIC COMPOUNDS
HORMONES
HYDROCARBONS
HYDROGEN COMPOUNDS
HYPNOTICS AND SEDATIVES
ISOTOPE APPLICATIONS
KINETICS
MAMMALS
MEMBRANE PROTEINS
NUCLEOTIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANIC OXYGEN COMPOUNDS
ORGANOIDS
ORGANS
POLYCYCLIC AROMATIC HYDROCARBONS
PYRIMIDINES
REACTION KINETICS
RIBOSOMES
RODENTS
STEROID HORMONES
VERTEBRATES
550501* - Metabolism- Tracer Techniques
560300 - Chemicals Metabolism & Toxicology