A comparative study on the irreversible binding of labeled halothane trichlorofluoromethane, chloroform, and carbon tetrachloride to hepatic protein and lipids in vitro and in vivo.
1) After intraperitoneal injection of labeled CCl4, CHCl3, and halothane in mice, 14C is preferentially bound to liver endoplasmic protein and lipid. A considerable activity is also associated with mitochondrial constituents. Maximal protein binding (nmol/mg): CCl4: 2.8 (0.5 hrs); CHCl3: 11.5 (6 hrs); halothane: 5 (6 hrs). Lipid binding: CCl4: 6.4 (5 min); CHCl3: 8 (4 hrs); halothane: 13.5 (2 hrs). The form of the binding curves in microsomal and mitochondrial protein and lipid differed with the individual haloalkanes. 2) The irreversible (covalent) binding of 14C from labeled haloalkanes in anaerobic suspensions of isolated rabbit liver microsomes and NADPH after 30 min was for protein (lipid) (nmol/mg): CCl4: 15 (58); CHCl3: 3.4 (3.2); halothane: 2.3 (10); trichlorofluoromethane: 6.5 (30). Anerobic incubation favored dehalogenation, but CHCl3 metabolism and irreversible binding requires oxygen. The greatest differences in the in vitro "covalent" binding rates were observed with CHCl3 in rat, mouse, and rabbit. 3) Altered microsomal cytochrome P-450 concentrations in newborn animals, or produced by pretreatment of rats with phenobarbital, 3-methylcholanthrene (MC), or CoCl2 effected similar, but not proportional changes in the rates of irreversible protein and lipid binding. Upon addition of CCl4 the difference of light absorption of reduced liver microsomes from MC-pretreated rats containing cytochrome P-448 appeared at 452 nm. The irreversible binding rate in these microsomes was also increased. The small accleration in irreversible binding in liver microsomes from rats pretreated with isopropanol is not proportional to the high increase of CCl4 toxicity. 4) Practically no binding to added, soluble albumin or RNA was observed in microsomal incubates. However, 14C is bound to the nicotine-adenine dinucleotides of the NADPH system. All haloalkanes produced a similar increase of NADPH oxidation in incubates of rabbit liver microsomes and NADPH.
A comparative study on the irreversible binding of labeled halothane trichlorofluoromethane, chloroform, and carbon tetrachloride to hepatic protein and lipids in vitro and in vivo. 1) After intraperitoneal injection of labeled CCl4, CHCl3, and halothane in mice, 14C is preferentially bound to liver endoplasmic protein and lipid. A considerable activity is also associated with mitochondrial constituents. Maximal protein binding (nmol/mg): CCl4: 2.8 (0.5 hrs); CHCl3: 11.5 (6 hrs); halothane: 5 (6 hrs). Lipid binding: CCl4: 6.4 (5 min); CHCl3: 8 (4 hrs); halothane: 13.5 (2 hrs). The form of the binding curves in microsomal and mitochondrial protein and lipid differed with the individual haloalkanes. 2) The irreversible (covalent) binding of 14C from labeled haloalkanes in anaerobic suspensions of isolated rabbit liver microsomes and NADPH after 30 min was for protein (lipid) (nmol/mg): CCl4: 15 (58); CHCl3: 3.4 (3.2); halothane: 2.3 (10); trichlorofluoromethane: 6.5 (30). Anerobic incubation favored dehalogenation, but CHCl3 metabolism and irreversible binding requires oxygen. The greatest differences in the in vitro "covalent" binding rates were observed with CHCl3 in rat, mouse, and rabbit. 3) Altered microsomal cytochrome P-450 concentrations in newborn animals, or produced by pretreatment of rats with phenobarbital, 3-methylcholanthrene (MC), or CoCl2 effected similar, but not proportional changes in the rates of irreversible protein and lipid binding. Upon addition of CCl4 the difference of light absorption of reduced liver microsomes from MC-pretreated rats containing cytochrome P-448 appeared at 452 nm. The irreversible binding rate in these microsomes was also increased. The small accleration in irreversible binding in liver microsomes from rats pretreated with isopropanol is not proportional to the high increase of CCl4 toxicity. 4) Practically no binding to added, soluble albumin or RNA was observed in microsomal incubates. However, 14C is bound to the nicotine-adenine dinucleotides of the NADPH system. All haloalkanes produced a similar increase of NADPH oxidation in incubates of rabbit liver microsomes and NADPH.