Covalent liver microsome

Roy, D. and Snodgrass W.R. 1990. Covalent binding of phenytoin to protein and modulation of phenytoin metabolism by thiols in A/J mouse liver microsomes. J. Pharmacol. Exp. Ther. 252 895. 

Cytochrome P-450 spectra (reduced + CO) show the 2-nm shift to the blue as a result of 3-MC induction. No such shift is observed in the trout, control rat, and PB-induced rat liver microsomes. Cytochrome P-450 EtNC spectra were recorded at pH 7.4, 2 min after the samples were reduced with dithionite. The absorption peaks are at 430 and 455 nm. For pH curves the A Absorbance represents A A (430—490 nm) and A A (455— 490 nm). The number of (+) signifies only the relative activity or inhibition with respect to BP hydroxylation and covalent binding of BP to DNA ( ++-(-) signifies 2-4 times and (+-H—h) signifies 10-36 times the control rat microsomal activity (n.d.), not determined. 

It has been noted that cell cultures derived from trout (66, 67) and trout liver microsomes (U2, U7, 68) may have relatively high AHH activity (BP hydroxylase) which sometimes exceeds the activity observed in control rat liver microsomes. The metabolite pattern obtained using trout liver microsomes resembled that produced by MC treated rat liver microsomes (P-Uh8)(6 and Ahokas, Saarni, Nebert and Pelkonen, manuscript in preparation Table IV). Associated with this pattern of BP metabolites was covalent binding to DNA which was three times as high as obtained by using rat liver microsomes. Another species of fish (roach), on the other hand was found to be almost inactive in catalyzing in vitro bind-... 

The in vitro metabolism and covalent binding of benzo(a) pyrene to DNA catalysed by trout liver microsomes. 

Typically, the extent of covalent binding of microsomal protein may be evaluated by incubating or -labeled dmg substance with liver microsomes in the presence... 

Zhao, S.X. et al. (2007) NADPH-dependent covalent binding of [3H] paroxetine to human liver microsomes and S-9 fractions identification of an electrophilic quinone metabolite of paroxetine. Chemical Research in Toxicology, 20 (11), 1649-1657. 

In a study in which liver microsomes, prepared from male Wistar rats (200-250 g), were incubated with 5 x 10 mol/L2,3-dibromopH]propan-l-olandanNADPH-gene-rating system, covalent binding to protein was determined. Addition of the epoxide hydrolase inhibitor l,l,l-trichloropropene-2,3-epoxide led to an increase in the proteinbinding rate of 2,3-dibromopropan-l-ol (Soderlund etal., 1981). 

An in vitro study with a C-PBB mixture containing 12 major components found only traces of radioactivity bound to rat liver microsomal macromolecules (Dannan et al. 1978a). Binding, however, was dependent upon the type of microsomes used to activate the PBB mixture. Microsomes isolated from animals pretreated with methylcholanthrene (MC) bound twice the amount of radioactivity compared with controls, whereas activation with phenobarbital (PB) or PBB bound 5 times more radioactivity than control microsomes. Also, the authors showed that no radioactivity was covalently bound to DNA following incubation with C-PBB. The type of microsomes used or the presence or... 

Thus, the Vmax for the covalent binding to protein is higher for the lung microsomal enzyme than for the liver, whereas the apparent Km for the lung enzyme is about one-tenth that for the liver microsomal enzyme preparation. The lung enzyme, therefore, has a greater affinity for the ipomeanol and activates it more rapidly. GSH inhibited the binding to protein in vitro. 

Phenol is converted by rat liver microsomes to a reactive metabolite that binds covalently to protein the most likely metabolites involved in this are hydroquinone and, at... 

Incubation of l,l,2,2,-tetrachloro[l,2- 4C]ethane with a reconstituted monooxygenase system or with intact rat liver microsomes led to the formation of a metabolite capable of binding covalently to proteins and other nucleophiles. The only soluble metabolite detected upon incubation of 1,1,2,2-tetrachloroethane with a reconstituted system was dichloroacetic acid. Pronase digestion of the C-labcllcd microsomal proteins indicated the presence of several derivatized amino acids, which were hydrolysed by alkali to yield dichloroacetic acid. The results are consistent with biotransformation of 1,1,2,2-tetrachloroethane by cytochrome P450 to dichloroacetyl chloride, which can bind covalently to various nucleophiles or hydrolyse to dichloroacetic acid (Halpert, 1982). 

In a general sense, the kinetics and metabolism of toluene in humans, rats and mice are very similar the hippurate is in all cases by far the major metabolite, while in all species the ortho- and / ora-cresols are minor metabolites. To what extent formation of a potentially reactive sulfate conjugate of benzyl alcohol occurs (van Doom et al., 1980 Chidgley et al., 1986) is uncertain, mainly because mercapturates formed from toluene have not been characterized. Similarly, whether the covalent binding observed in rat liver microsomes has any toxicological relevance is uncertain. 

BIT, Binding (covalent) to microsomal proteins, Wistar rat liver and NT + 43.5 Soderlund etal. (1981)... 

Less than 0.07% of the recovered urinary radioactivity in rats given 100 or 1000 mg/kg bw [ Clacetamide coeluted upon high-performance liquid chromatography with an N-hydroxyacetamide standard and this hydroxamic acid could not be detected after incubation of acetamide with rat liver microsomes and NADPH or in primary cultures of rat hepatocytes. [ C]Acetamide does not bind covalently to proteins in the presence of rat liver microsomes and NADPH or cytosolic fraction, whereas hepatocyte cultures contained non-extractable radioactivity. This association was inhibited by cycloheximide to the same extent as [ CJacetate incorporation into cellular proteins (Dybing et al., 1987). 

Male Fischer 344 rats were exposed by inhalation to 1% 2-chloro-1,1,1 -trifluoroethane for 2 h and then urine was collected for 24 h. Urinary metabolites identified by 19F nuclear magnetic resonance and gas chromatography/mass spectrometry were 2,2,2-trifluoroethyl glucuronide (16%), trifluoroacetic acid (14%), trifluoroacetaldehyde hydrate (26%), trifluoroacetaldehyde-urea adduct (40%) and inorganic fluoride (3%). A minor, unidentified metabolite was also detected. No covalent binding of fluorine-containing metabolites was observed in the liver and kidney from the exposed rats (Yin et al., 1995). In-vitro incubation of 2-chloro-1,1,1-trifluoroethane with rat liver microsomes and an NADPH-generating system has been shown to involve a dechlorination reaction (Salmon et al., 1981) that produced trifluoroacetaldehyde hydrate as the only metabolite (Yin et al., 1995). 

Obach, R. S., Kalgutkar, A. S., Soglia, J. R., and Zhao, S. X. (2008). Can in vitro metabolism-dependent covalent binding data in liver microsomes distinguish hepatotoxic from non-hepatotoxic drugs An analysis of 18 drugs with consideration of intrinsic clearance and daily dose. Chem. Res. Toxicol. 21 1814-1822. 

Madan A, Parkinson A. Characterization of the NADPH-dependent covalent binding of [14-C]-halothane to human liver microsomes a role for CYP2E1 at low substrate concentrations. Drug Metab Dispos 1996 24 1307-1313. 

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