Use of heterologously-expressed cytochrome P450 and glutathione transferase enzymes in toxicity assays.
Our groups have had a long-term interest in utilizing bacterial systems in the characterization of bioactivation and detoxication reactions catalyzed by cytochrome P450 (P450) and glutathione transferase (GST) enzymes. Bacterial systems remain the first choice for initial screens with new chemicals and have advantages, including high-throughput capability. Most human P450s of interest in toxicology have been readily expressed in Escherichia coli with only minor sequence modification. These enzymes can be readily purified and used in assays of activation of chemicals. Bicistronic systems have been developed in order to provide the auxiliary NADPH-P450 reductase. Alternative systems involve these enzymes expressed together within bacteria. In one approach, a lac selection system is used with E. coli and has been applied to the characterization of inhibitors of P450s 1A2 and 1B1, as well as in basic studies involving random mutagenesis. Another approach utilizes induction of the SOS (umu) response in Salmonella typhimurium, and systems have now been developed with human P450s 1A1, 1A2, 1B1, 2C9, 2D6, 2E1, and 3A4, which have been used to report responses from heterocyclic amines. S. typhimurium his reporter systems have also been used with GSTs, first to demonstrate the role of rat GST 5-5 in the activation of dihalomethanes. These systems have been used to compare these GSTs with regard to activation of dihaloalkanes and potential toxicity.
Guengerich
F Peter
FP
Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building, Medical Research Building I, Nashville, TN 37232-0146, USA. guengerich@toxicology.mc.vanderbilt.edu
Wheeler
James B
JB
Chun
Young-Jin
YJ
Kim
Donghak
D
Shimada
Tsutomu
T
Aryal
Pramod
P
Oda
Yoshimitsu
Y
Gillam
Elizabeth M J
EM
D016588:Anticarcinogenic Agents / Q000494:pharmacology; D001419:Bacteria / Q000187:drug effects / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D004247:DNA / Q000187:drug effects; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005982:Glutathione Transferase / Q000096:biosynthesis*; D006801:Humans; D009153:Mutagens / Q000633:toxicity*; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics; D014116:Toxicology / Q000379:methods*
D016588:Anticarcinogenic Agents; D018076:DNA, Complementary; D009153:Mutagens; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; D005982:Glutathione Transferase