Effects of elevated temperature on misonidazole O-demethylation by mouse liver microsomes: kinetic and stability studies of a model mixed-function oxidase reaction.
We investigated the effects of a range of temperatures (33 degrees-44 degrees C) on the stability and kinetics of C3H mouse liver microsomal misonidazole (MISO) O-demethylase in vitro. Microsomal O-demethylase activity was stable for 60 min at 37 degrees C and for 30 min at 41 degrees C but was steadily inactivated with longer incubation times. Inactivation at 44 degrees and 47 degrees C was exponential, with half-lives of 41 and 11 min, respectively. MISO O-demethylation followed Michaelis-Menten kinetics from 33 degrees to 44 degrees C. The apparent Vmax for desmethylmisonidazole (Ro 05-9963) formation was decreased by 32% (from 2.14 to 1.47 nmol min-1 mg-1 protein) with a 4 degrees decrease from 37 degrees to 33 degrees C. An increase of 4 degrees from 37 degrees to 41 degrees C enhanced the Vmax by 47%, but there was only an additional 9% increase for a further 3 degrees rise to 44 degrees C. Apparent Km values were unaltered at about 1.6 mM. These results show that elevated temperatures in the clinically relevant hyperthermia range (41 degrees-44 degrees C) can enhance a model mixed-function oxidase reaction in vitro. Such effects may be important for the metabolism, activity and toxicity of anticancer drugs combined with hyperthermia in vivo.
Walton
M I
MI
MRC Clinical Oncology and Radiotherapeutics Unit, Cambridge, UK.
Bleehen
N M
NM
Workman
P
P
D000818:Animals; D002384:Catalysis; D002851:Chromatography, High Pressure Liquid; D004795:Enzyme Stability; D066298:In Vitro Techniques; D008297:Male; D008745:Methylation; D051379:Mice; D008862:Microsomes, Liver / Q000201:enzymology*; D008920:Misonidazole / Q000031:analogs & derivatives / Q000032:analysis / Q000493:pharmacokinetics*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010084:Oxidation-Reduction; D010090:Oxidoreductases, O-Demethylating / Q000378:metabolism; D013696:Temperature; D013997:Time Factors
D008920:Misonidazole; D006899:Mixed Function Oxygenases; D010090:Oxidoreductases, O-Demethylating; C009997:desmethylmisonidazole