Isoflurane and cytochrome b5 stimulation of 2-chloro-1,1-difluoroethene metabolism by reconstituted rat CYP2B1 and CYP2C6.

Isoflurane stimulates the metabolism of 2-chloro-1,1-difluoroethene (CDE) in liver microsomes from phenobarbital-treated rats or rabbits. The P450 isozymes involved and the mechanism by which such stimulation occurs have not been clarified. The present study examined the effects of isoflurane and cytochrome b5 on CDE metabolism in reconstituted systems containing purified rat CYP2B1 or CYP2C6. Under similar incubation conditions, CYP2B1 defluorinated CDE at approximately five times the rate of CYP2C6. Isoflurane was a potent stimulator of CDE metabolism, increasing it nearly 5-fold when catalyzed by CYP2B1, but only 2-fold when catalyzed by CYP2C6. Isoflurane had no stimulatory effect on benzphetamine metabolism by CYP2B1 or CYP2C6. Cytochrome b5 was not required for isoflurane-facilitated CDE metabolism; however, the addition of cytochrome b5 to CYP2B1 increased CDE metabolism 71 and 44%, in the absence and presence of isoflurane, respectively. In reconstituted CYP2B1, isoflurane generated a type I difference spectrum of approximately twice the magnitude of CDE and stimulated NADPH consumption more so than CDE. The same quantity of NADPH was consumed when CDE was present with isoflurane as compared with isoflurane alone. These data support the hypothesis that isoflurane stimulates CDE metabolism by a mechanism involving increased P450 reduction via direct isoflurane interaction with P450.

Effects of diabetes on calcium uptake by rat brush border membrane vesicles.

1. We investigated the mechanism of decreased transmucosal calcium transport in the gut of the diabetic rat by comparing calcium uptake by brush border membrane vesicles from control and streptozotocin diabetic rats at 5 days. Brush border calcium uptake consists of saturable and non-saturable components. Saturable uptake is mediated by a specific mobile carrier mechanism and is defined by Vmax (saturable uptake of calcium at infinite medium calcium concentration) and KT (calcium concentration at Vmax/2). Non-saturable uptake is defined by kD (rate constant for non-saturable uptake per unit calcium concentration), and comprises both diffusive and surface binding components of calcium uptake. 2. We found both saturable and non-saturable calcium uptake to be decreased (P < 0.05) in diabetes. Comparing control and diabetic, Vmax was 247 compared to 152 (data are pmol/mg protein per 3 s); kD was 285 compared to 172 (data are pmol/mg protein per 3 s at 1 mmol/L calcium); and KT (mmol/L) did not differ between groups, 0.070 compared to 0.057. 3. The decreased Vmax in the setting of unchanged KT in vesicles from diabetics is consistent with decreased calcium transporter specific activity, rather than with altered transporter function. 4. Since (i) Vmax is decreased by vitamin D deficiency in the normal rat, and (ii) circulating 1 alpha, 25-dihydroxycholecalciferol is decreased in the diabetic rat, decreased Vmax in the diabetic may be related to the low 1 alpha,25-dihydroxycholecalciferol.(ABSTRACT TRUNCATED AT 250 WORDS)

Isoflurane-chlorodifluoroethene interaction in human liver microsomes. Role of cytochrome P4502B6 in potentiation of haloethene metabolism.

Short-chain saturated halocarbons, including isoflurane and the chlorofluorocarbon substitute HCFC-123, can strongly potentiate the cytochrome P450-dependent oxidation of gaseous haloethenes, such as 2-chloro-1,1-difluoroethene (CDE) and vinyl chloride, in vivo and in vitro. P450 isozyme specificity in this effect is suggested by the fact that the interaction is pronounced in microsomes from rats treated with phenobarbital, but does not occur in microsomes of isoniazid- or beta-naphthoflavone-treated animals. We examined the effect of isoflurane on CDE defluorination in liver microsomes from 10 human organ donors to determine whether saturated halocarbon/haloethene interactions also occur in humans and, if so, to determine the cytochromes P450 involved. Three of the samples exhibited isoflurane-stimulated increases (24, 32, and 41%) in CDE defluorination; isoflurane either inhibited or had no effect on CDE metabolism in the other seven samples. Two samples in which isoflurane potentiated CDE metabolism to the greatest rates had higher coumarin 7-hydroxylase (indicative of CYP2A6), 7-ethoxycoumarin O-deethylase (CYP2B6), and nifedipine oxidase (CYP3A4) activities than the other eight samples. However, all 10 subjects had similar rates of phenacetin O-deethylation (CYP1A2) and chlorzoxazone 6-hydroxylation (CYP2E1). In microsomes from cells transfected with cDNAs coding for individual human P450s, CDE metabolism by CYP2B6 was stimulated (216%) by isoflurane, whereas isoflurane did not stimulate CDE metabolism by human CYP2A6, CYP3A4, CYP2D6, or CYP2E1. Isoflurane highly increased CDE defluorination in purified rat CYP2B1 (470%).(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory effects of propofol on cytochrome P450 activities in rat hepatic microsomes.

The effects of propofol on cytochrome P450 activity in rat hepatic microsomes were evaluated to determine the potential influence of this anesthetic on the metabolism of coadministered agents. In microsomes from untreated and isoniazid-treated rats, propofol was a weak inhibitor of enflurane metabolism, inhibiting activity only at 0.35 mM propofol. In contrast, toluene, a related compound, effectively impaired enflurane defluorination in microsomes from untreated, and isoniazid- and phenobarbital-treated rats at concentrations as low as 0.025 mM. Propofol, in contrast to toluene, was an effective inhibitor of benzphetamine demethylation where it inhibited this activity at propofol concentrations as low as 0.025 mM in microsomes from phenobarbital-treated rats. In microsomes from phenobarbital-treated rats, propofol potently inhibited the metabolism of aniline. Sixty-four percent inhibition was achieved at 0.03 mM propofol, whereas toluene had no effect at 1 mM. These data demonstrate that propofol does not effectively inhibit enflurane metabolism performed by the isoniazid-inducible cytochrome P450IIE1 but effectively impairs activities of the phenobarbital-inducible cytochrome P450 isozymes.

Contrasting effects of 1,1,1-trichloroethane on [14C]vinyl chloride metabolism and activation in hepatic microsomes from phenobarbital- and isoniazid-treated rats.

The interaction of 1,1,1-trichloroethane (TCE), a widely used chlorocarbon solvent, on the metabolism and activation of [14C]-vinyl chloride in rat hepatic microsomes was investigated to determine the effects of combined exposures to these compounds. In microsomes from phenobarbital (PB)-treated rats, TCE increased vinyl chloride-protein binding and vinyl chloride aqueous metabolite formation over twofold when vinyl chloride 0.32% (v/v) and TCE (0.65%) are incubated together. In contrast, under similar incubation conditions, TCE inhibited vinyl chloride metabolism and protein binding up to 45% in microsomes from isoniazid-treated animals. TCE also inhibited vinyl chloride metabolism and binding in microsomes from untreated rats, but to a lesser degree. Like the effect of TCE on vinyl chloride-protein binding, TCE increased vinyl chloride binding to DNA approximately 130% in microsomes from PB-treated rats, yet inhibited vinyl chloride-DNA binding in microsomes from isoniazid-treated and untreated animals. Consistent with TCE effects on vinyl chloride binding and aqueous metabolite production, TCE further increased cytochrome P450 loss due to vinyl chloride metabolism in microsomes from PB-treated rats, but was inhibitory to cytochrome P450 loss due to vinyl chloride metabolism in microsomes from isoniazid-treated and untreated rats. These data demonstrate that the relatively metabolically inert solvent, 1,1,1-trichloroethane, can directly increase vinyl chloride metabolism and activation catalyzed by the phenobarbital-inducible isozymes, but is inhibitory toward vinyl chloride metabolism catalyzed by the isoniazid-inducible CYP2E1.

Acute stimulation of trifluoroethene defluorination and cytochrome P450 inactivation in the rat by exposure to isoflurane.

The effect of isoflurane on trifluoroethene (TFE) defluorination and cytochrome P450 inactivation was examined in rats to determine the influence of this anesthetic on in vivo fluoroethene metabolism. Exposure of rats to TFE (0.5%, v/v) or a mixture of TFE and isoflurane (0.5% each) in oxygen for 60 min resulted in plasma fluoride increased over that in nonexposed or isoflurane (0.5%)-exposed animals. In untreated rats plasma fluoride levels following TFE and TFE-isoflurane exposures were approximately equal. In rats treated with phenobarbital, however, isoflurane increased plasma fluoride over two times that in rats exposed to TFE alone. Likewise cytochrome P450 levels declined 24% in TFE-exposed animals and 64% in rats exposed to TFE-isoflurane. The ability of microsomes from fluorocarbon-exposed animals to metabolize (R)- and (S)-warfarin indicates that TFE exposure inactivated the phenobarbital-inducible isozymes P450IIB1, P450IIC6, and P450IIIA to approximately equal degrees (21-35%). TFE-isoflurane exposure further inhibited P450IIB1 and PB450IIC6 to 50-70%, but had only a minor effect on P450IIIA activity. These data demonstrate that the defluorination of TFE in vivo by the phenobarbital-inducible cytochrome P450 isozymes is increased by isoflurane, and that isoflurane enhances the ability of TFE to inactivate cytochromes P450 in an isozyme-selective manner.