Browsing by Author "Bradshaw, Jennifer Jean"

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    Isoflurane : interaction with hepatic microsomal enzymes
    (1992) Bradshaw, Jennifer Jean; Gevers, Wieland; Ivanetich, Kathryn M
    lsoflurane interacts with cytochrome P-450 in rat and human hepatic microsomes and the Δ6- and Δ5-desaturases in rat hepatic microsomes. The interaction of isoflurane with cytochrome P-450 results in its metabolism to fluoride ion and organofluorine metabolites. The cytochrome P-450 isozymes catalysing the defluorination of isoflurane were assessed in hepatic microsomes from phenobarbital-, β-naphthoflavone- and pregnenolone-16α-carbonitrilepretreated and untreated rats. One or more of the cytochrome P-450 isozymes induced by phenobarbital and pregnenolone-16α-carbonitrile appear to defluorinate isoflurane, but those induced by β-naphthoflavone do not. From a comparison of the extent of defluorination of isoflurane in hepatic microsomes from phenobarbital- and pregnenolone-16α-carbonitrile-pretreated rats, and their Kₘ and Vₘₐₓ values, it appears that isoflurane is defluorinated by one or more isozymes induced by both phenobarbital and pregnenolone-16α-carbonitrile. The major isozyme is probably cytochrome P-450PCN1. The metabolites of isoflurane were identified in human and phenobarbital-induced rat hepatic microsomes. In microsomes from phenobarbital-pretreated rats, isoflurane is metabolised to fluoride ion and trifluoroacetaldehyde; trifluoroacetic acid is not produced in measureable amounts. The trifluoroacetaldehyde produced binds to microsomal constituents. In human hepatic microsomes, the organofluorine metabolite is identified as trifluoroacetic acid. It is proposed that isoflurane is metabolised by different pathways in human and phenobarbital-induced rat hepatic microsomes. The interaction of isoflurane with the cyanide-sensitive factors was assessed by several criteria. Firstly, using the reoxidation of cytochrome b₅ as an index of fatty acid desaturase activity, isoflurane appears to interact with the Δ6- and/or Δ5-desaturases, but not the Δ9-desaturase. Secondly, these results were confirmed and clarified by the use of direct assays to measure the fatty acid desaturase activity. Using the direct assay, we confirmed that isoflurane did not inhibit the Δ9-desaturase and inhibited Δ6-desaturation of linoleic acid, but not the Δ6-desaturation of α-linolenic acid. The inhibition of the Δ6-desaturation of linoleic acid occurred at low millimolar concentrations of isoflurane. lsoflurane inhibits the Δ5-desaturation of eicosa-8, 11, 14-trienoic acid to a small extent which is only apparent at much higher concentrations of isoflurane than that which inhibits the Δ6-desaturase. Further studies focussed on measurement of the activity of Δ6-desaturase in order to attempt to study the kinetics of the inhibition of the Δ6-desaturase by isoflurane: Δ6-desaturase activity was assessed using hepatic microsomes as the source of the enzyme and linoleic acid as substrate precursor. In the course of these studies, we identified a number of factors that affected the apparent activity of the Δ6-desaturase in hepatic microsomes. These included significant levels of endogenous substrate and competing reactions in the hepatic microsomes. Endogenous substrate levels were quantified and corrected for. We then resorted to computer modelling to extract the kinetics of the Δ6-desaturase free of contributions from acyl-CoA synthetase and lysophospholipid acyltransferase, as well as enzyme decay. The kinetics of isoflurane inhibition of the Δ6-desaturase were then superimposed and studied by computer modelling.
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    The fluroxene mediated degradation of cytochromes P-450
    (1977) Bradshaw, Jennifer Jean; Ivanetich, Kathryn M.
    The degradation of cytochromes P-450 by fluroxene (2,2,2-trifluoroethyl vinyl ether) has been investigated. Fluroxene is shown to specifically degrade cytochromes P-450 in vivo and in vitro without affecting the levels of the other microsomal enzymes, cytochrome ~S a.nd NADPH-cytochrome~ reductase. Fluroxene appears to degrade the haem moiety of cytochromes P-450 but does not affect the level of the apoprotein. The degradation of cyto-chromes P-450 by fluroxene is accompanied by a loss of E-nitroanisole 0-demethylase and biphenyl 4-hydroxylase activities and a decrease in the extent of aniline binding is observed. By using cytochromes P-450 dependent reactions which are catalysed by specific type P-450 cytochromes,~.~· the hydroxylation of benzpyrene, the N-demethylation of ethyl-morphine and the binding of ethyl isocyanide, it is established that only cytochrome P-450 is degraded by fluroxene in vivo following phenobarbital induction of animals, and both cytochrome P-450 and cytochrome P-448 following methylcholanthrene induction. The same type P-450 cytochromes are shown to be degraded by fluroxene in vitro in phenobarbital and methylcholanthrene induced microsomes. This was established from studies of the kinetics of the fluroxene mediated degradation of cyto-chromes P-450. In addition, the K values for the flurox-m ene mediated degradation of cytochromes P-450 differ with iii the different inducing agents and indicate the involve-ment of two different type P-450 cytochromes in the degradation reaction in methylcholanthrene induced micro-somes. Metabolic activation of cytochromes P-450 by the cyto-chromes P-450 drug metabolising pathway appears to be essential for the fluroxene mediated degradation of cyto-chromes P-450. Since none of the known or proposed metabolites of fluroxene can mimic the degradation of cytochromes P-450 by fluroxene, a reactive species is proposed to be involved. By varying the experimental conditions, and with the use of inhibitors of cytochromes P-450, the likely sequence of events in the fluroxene mediated degradation of cytochromes P-450 is shown to be as follows: fluroxene is metabolised by cytochrome P-450 to a transient reactive intermediate which has the ability to degrade the haem moiety of cytochrome P-450 and cyto-chrome P-448. By comparing the ability of various analogues of fluroxene to degrade cytochromes P-450, it is established that the formation of the proposed reactive intermediate is dependent on the presence of the vinyl moiety of the molecule. Initial studies indicate that the reactive species may take the form of an epoxide.