Visualization of a covalent intermediate between microsomal epoxide hydrolase, but not cholesterol epoxide hydrolase, and their substrates

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of alpha/beta-hydrolase-fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme-substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with [1-(14)C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein-bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1,1,1-trichloropropene oxide in the incubation mixture. In a similar manner, 4-fluorochalcone-oxide-sensitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26-(14)C]cholesterol-5alpha,6alpha-epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme-substrate-ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.     

Effects of dithiocarbamates on toxicity of cadmium in rat primary hepatocyte cultures

The protective effects of N-benzyl-D-glucamine dithiocarbamate (BGD) and N-p-hydroxymethylbenzyl-D-glucamine dithiocarbamate (HBGD) on the toxicity of Cd in the rat primary hepatocyte cultures were studied. Cytotoxicity was assessed by measuring cell viability, extra cellular lactic dehydrogenase (LDH) activity, and intracellular lipid peroxidation and active oxygen species. Primary hepatocyte cultures were treated with 109CdCl2 (5, 10 or 50 microM Cd and 1.7 KBq of 109Cd/well) for 30 min or 4 h. BGD or HBGD was added to the culture medium to make the final concentration of 100 microM and incubated for 4.5 h in 30 min Cd exposure or 1 h in 4 h Cd exposure. Decreases in the hepatocyte viability caused by all Cd exposure concentrations were significantly prevented by treatment with BGD or HBGD. The treatment with the chelating agents for 4.5 h after Cd exposure for 30 min significantly prevented increases in extracellular LDH activity. Increases in the lipid peroxidation in hepatocytes exposed to Cd for 30 min or 4 h were prevented significantly by treatment with BGD or HBGD for 4.5 h or 1 h, respectively. Moreover, the increases in the level of active oxygen species caused by Cd exposure for 30 min were significantly prevented by treatment with the chelating agents for 1.5 h. These findings suggest that BGD and HBGD protect against the cytotoxicity of Cd in rat primary hepatocyte cultures and that the protective effects of chelating agents presumably result from a decrease in the Cd level, the effective sequestration of the reactive Cd ion, and the direct preventive effect on the active oxygen species in the hepatocytes.     

Enhancement of radiation-induced hepatic microsomal epoxide hydrolase gene expression by oltipraz in rats

The effects of radiation exposure in conjunction with oltipraz, a chemopreventive agent, on the expression of the gene encoding hepatic microsomal epoxide hydrolase (mEH) were examined in rats. Rats exposed to a single dose of 3 Gy gamma rays exhibited timerelated changes in the hepatic mEH mRNA level. Whereas the mEH mRNA level was transiently decreased at 3 and 8 h after irradiation, the mRNA levels were increased 3- to 4-fold at 15 to 48 h postirradiation, returning to the level in untreated animals at 72 h. Treatment of rats with oltipraz resulted in 1- to 19-fold increases in hepatic mEH mRNA levels 24 h post-treatment at doses of 5-200 mg/kg. Although treatment with oltipraz at a dose of 30 mg/kg affected the mEH mRNA level minimally (i.e. approximately 2-fold), 3 Gy whole-body irradiation along with oltipraz treatment resulted in a 9-fold increase in the mEH mRNA level at 24 h post-treatment. Treatment of animals with both oltipraz and 3 Gy gamma radiation for 3 consecutive days resulted in a 7-fold increase in mEH mRNA, showing that the increases in mEH mRNA were enhanced by the combination treatment. In rats irradiated with 3 Gy for 5 consecutive days, however, the mEH mRNA level failed to increase due to cell injury. Studies were further designed to assess the effects of 0.5 Gy ionizing radiation and concomitant oltipraz treatment. RNA blot analysis showed that mEH mRNA levels failed to be significantly altered at 3, 8, 15, 24 and 48 h after a single dose of 0.5 Gy. Nonetheless, exposure of animals to 0.5 Gy daily for 3 to 5 consecutive days caused a 3-fold elevation in the hepatic mEH mRNA level. Furthermore, treatment of animals with both oltipraz (30 mg/kg/day) and 0.5 Gy of gamma rays resulted in an enhanced elevation in the mEH mRNA level at 24 h post-treatment compared to the individual treatment, resulting in a 7-fold relative increase. The enhanced expression of hepatic mEH mRNA by 0.5 Gy gamma radiation and oltipraz was also observed after treatment for 3 to 5 days (8- to 6-fold relative increases). Western immunoblot analyses showed that hepatic microsomes produced from the rats treated with 0.5 Gy daily for 3 to 5 days resulted in a approximately 2-fold induction of hepatic mEH and that rats exposed to radiation in combination with oltipraz showed 3-fold increases in the liver mEH protein. Thus the relative increase in mEH mRNA levels was consistent with the expression of the protein. These results demonstrate that ionizing radiation causes alterations in hepatic mEH gene expression with the induction of the protein and that the mEH gene expression is enhanced by oltipraz treatment.     

Induction of microsomal epoxide hydrolase activity in inbred mice by chronic phenytoin exposure

A lower microsomal epoxide hydrolase (mEH) activity has been associated with increased likelihood of fetal hydantoin syndrome. While phenytoin anticonvulsive regimens are long-term, there are no data regarding induction of mEH by chronic phenytoin exposure. Two inbred mouse strains which differ in their susceptibility (A/J > C57BL/6J) to phenytoin-induced oral clefting were treated with an oral gavage of phenytoin for 14 consecutive days. The mice were sacrificed on the 15th day, and hepatic microsomes were prepared. mEH activity was determined using benzo[a]pyrene-4,5-oxide. The dihydrodiol product was separated by HPLC and quantified. There was no significant difference (P = 0.15) in the phenytoin plasma level between the two strains on Day 15. There was no significant difference (P = 0.07) between control and sham control groups within each strain, so they were combined for further analysis. There was a significant strain difference (P = 0.0001) between the control and phenytoin-exposed group means, with the C57BL/6J strain having the greater activity before and after phenytoin exposure. The A/J phenytoin-exposed group activity was 51% higher (P = 0.01) than the A/J control, while the C57BL/6J phenytoin-exposed group activity was 78% higher (P = 0.001) than the C57BL/6J control. The greater mEH activity in the phenytoin-induced clefting resistant strain (C57BL/6J) before and after phenytoin exposure is consistent with a putative oxidative metabolism mechanism of phenytoin teratogenecity. Chronic phenytoin exposure induced mEH activity in both strains, although the strain with the greater enzyme activity prior to the exposure continued to have the greater activity following induction.     

Micronucleus assays using cytochalasin-blocked MCL-5 cells, a proprietary human cell line expressing five human cytochromes P-450 and microsomal epoxide hydrolase

A total of 100 S. hyicus strains isolated from healthy piglets and piglets with exudative epidermitis originating from 100 different herds was examined for drug-resistance and prevalence of plasmids. Resistance to macrolide/linosamide antibiotics could be related to plasmids in 55 (93%) of the 59 resistant strains: A plasmid of 2.4 kb mediating resistance to macrolides and lincosamides was observed in 25 strains, and a plasmid of 11.5 kb mediating resistance to both macrolides/lincosamides and tetracycline was observed in 30 strains. A plasmid with a molecular weight of 4.5 kb was shown by curing experiments to be associated with resistance to tetracycline in 12 strains. All together, 47 strains were resistant to tetracycline. In 42 (89%) of these strains tetracycline-resistance was found to be encoded by plasmids. Fifty six strains were resistant to streptomycin, and resistance was associated with the presence of a 4.4 kb plasmid in 17 strains studied. Resistance to penicillin, observed in 44 strains, and resistance to kanamycin, observed in 15 strains, could not be related to plasmids in any of these strains. The 11.5 kb plasmid was observed in 39% of the strains isolated from piglets with EE, and in 7% of the strains isolated from healthy piglets. Despite its higher prevalence in strains from piglets with EE, the 11.5 kb plasmid could not be shown to encode production of capsule or exfoliative substances: factors which might play a role in the development of exudative epidermitis in piglets.     

cDNA cloning and expression of a soluble epoxide hydrolase from human liver

We report the cloning and expression of a cDNA that encodes a soluble epoxide hydrolase from human liver. The 2101-base clone predicts a 554-residue protein (M(r) 62,640) with an apparently imperfect peroxisomal targeting signal of Ser-Lys-Met at the carboxy terminus. The cDNA was expressed in the baculovirus system in the Spodoptera frugiperda 21 cell line. The recombinant protein was similar to soluble epoxide hydrolase isolated from human liver in terms of molecular weight, hydrolytic activity, inhibition, and immunoreactivity.     

Interactions between metallothionein inducers in rat liver and primary cultures of rat hepatocytes

The interaction of Zn, stress and endotoxin on liver metallothionein (MT) regulation has been studied in the rat. Zn, stress and endotoxin increased liver MT levels significantly, by 12-, 5- and 8-fold, respectively. The previous administration of Zn to stress or endotoxin treatments increased MT levels by 35- and 42-fold, respectively, indicating a synergistic effect in both cases. In contrast, when liver MT was preinduced by stress, MT levels were further increased by endotoxin only in an additive manner. In another experiment where liver MT induction by stress was studied in control rats and in rats with preinduced MT by Zn, endotoxin or stress, it was found that Zn pretreated animals had higher MT-I mRNA levels than endotoxin- or stress-pretreated ones. No synergisms between dexamethasone, Zn, TNF and IFN were observed in primary culture of hepatocytes. These results suggest that the observed synergisms between Zn and other MT inducers in vivo in the liver is a consequence of increased Zn levels in the body and mobilization capacity, with concomitant MT synthesis.     

Lipopolysaccharide inhibition of rat hepatic microsomal epoxide hydrolase and glutathione S-transferase gene expression irrespective of nuclear factor-kappaB activation

Lipopolysaccharide (LPS) is an endotoxin involved in septic shock syndrome and potentiates toxicant-induced liver injury. The effects of LPS on the constitutive and inducible expression of hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) genes were studied in rats. Northern blot analysis showed that treatment of rats with LPS caused suppression in mEH and GST gene expression. The mEH mRNA level was decreased in a time-dependent manner following a single dose of LPS (1 mg/kg, i.v.), resulting in levels of 52%, 22%, 17%, and 94% of those in untreated animals at 2, 6, 12, and 24 hr, respectively. The levels of rGSTA2 and rGSTA3 mRNA were suppressed in response to an LPS injection to the similar extents as observed in mEH mRNA, whereas rGSTM1 and rGSTM2 mRNA levels were less affected. LPS inhibited mEH gene expression at the doses of 1 microg or greater. Whereas treatment of rats with allyl disulfide (ADS), oltipraz (OZ) or pyrazine (PZ) at the dose of 50 mg/kg caused increases in the mEH mRNA level at 12 hr, a concomitant LPS injection (1 mg/kg) resulted in 80%-95% suppression of the inducible gene expression. The inducible rGSTA2, rGSTA3, rGSTM1, and rGSTM2 mRNA levels were also 50%-90% decreased at 12 hr after LPS treatment, with the relative change in rGSTA being greater than that in rGSTM. Three consecutive daily treatments with LPS (10 microg/kg/day) resulted in significant decreases of the constitutive and PZ (50 mg/kg/day, i.p. for 3 days)-inducible mEH and GST mRNA levels, which were consistent with those in the protein levels. Gel shift retardation analysis showed that LPS substantially activated the hepatic nuclear p65/p50 nuclear factor-kappaB (NF-kappaB) complex with the maximal effect observed at 1 hr at the doses of 1 microg/kg or greater. LPS-induced activation of nuclear NF-kappaB (1 microg/kg, i.v.) failed to be inhibited by concomitant treatment with the mEH and GST inducers, including ADS (300 mg/kg, p.o.), OZ (300 mg/kg, p.o.), and PZ (300 mg/kg, i.p.), indicating that NF-kappaB activation was not required for suppression of the gene expression by LPS. In contrast, GdCl3, an inhibitor of mEH and GST expression, inhibited LPS-induced activation of the p65/p50 NF-kappaB. These gel shift analyses provided evidence that LPS-induced activation of the NF-kappaB was not responsible for alterations in the gene expression. In summary, the results of this research demonstrate that LPS effectively inhibits constitutive and inducible mEH and GST expression with decreases in their mRNA levels, and that LPS suppression in the expression of the detoxifying enzymes is not mediated with its activation of NF-kappaB.     

Effects of Helicobacter pylori vacuolating cytotoxin on primary cultures of human gastric epithelial cells

BACKGROUND: Many Helicobacter pylori strains produce a cytotoxin that induces cytoplasmic vacuolation in various types of eukaryotic cells. In contrast with the marked cell vacuolation that occurs in vitro in response to this cytotoxin, comparatively little epithelial vacuolation has been observed in the gastric mucosa of H pylori infected persons. AIMS: Experiments were performed to determine the susceptibility of human gastric epithelial cells in vitro to H pylori vacuolating cytotoxin activity. METHODS: Human gastric epithelial cells, harvested from upper gastrointestinal endoscopic biopsy specimens, were incubated overnight with broth culture supernatants from either a wild type cytotoxin producing (tox+) H pylori strain or an isogenic mutant strain that lacks cytotoxin activity. RESULTS: Prominent cytoplasmic vacuolation occurred in response to tox+ supernatant, but not supernatant from the isogenic mutant strain. Primary human gastric epithelial cells were significantly more sensitive to H pylori vacuolating cytotoxin activity than were either HeLa or AGS cells. Exposure of human gastric epithelial cells to high concentrations of tox+ supernatant for 48 hours caused lethal cell injury. CONCLUSIONS: These studies indicate that primary human gastric epithelial cells are highly sensitive to H pylori vacuolating cytotoxin activity.     

Microsomal epoxide hydrolase polymorphism and risk of spontaneous abortion

Fibrinopeptide B (M(r) 1552.58) was employed as a calibration compound for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) post-source decay (PSD) fragment ion analysis in the negative mode. Experiments were performed by using both continuous and delayed extraction, with the maximum reflectron voltages being 30 and 21 kV, respectively. For comparison, a common positive ion PSD calibrant, ACTH(18-39) (M(r) 2466.7), was also employed with positive ion calibration constants being applied to negative ion spectra. Using fibrinopeptide B as the calibrant, the negative ion PSD results for angiotensin II (M(r) 1046.2), renin substrate tetradecapeptide (horse) (M(r) 1759.0), and the custom-synthesized peptide (K2G4)2 (M(r) 987.1) showed a factor of 1.5-2 improvement in absolute mass accuracy. Typical absolute mass-to-charge ratio accuracies were within +/- 1 Thompson and were achieved even when the peptide being analyzed was more massive than fibrinopeptide B. In addition, both calibrants showed increased accuracy when experiments were conducted in the delayed extraction mode. Other advantages of using fibrinopeptide B are its moderate cost and the ability to perform calibration and sample analysis for negative ion PSD under the same instrumental conditions.     

Effects of curcumin on P-glycoprotein in primary cultures of rat hepatocytes

Curcumin is a natural phenolic compound found in the rhizomes of Curcuma longa and endowed with beneficial biological activities including antioxidant, anticarcinogenic and hepatoprotective effects. In this study curcumin was tested for its potential ability to interact in vitro with hepatic P-glycoprotein (Pgp), in a model system represented by primary cultures of rat hepatocytes, in which spontaneous overexpression of multidrug resistance (mdr) genes occurs. In both freshly-plated hepatocytes, containing low levels of Pgp, and 72 hour-cultured hepatocytes, containing high levels of Pgp, the Rhodamine-123 (R-123) efflux, which represents a specific functional test for Pgp-mediated transport, was inhibited by curcumin in a dose-dependent manner. Western blot analysis showed that 25microM curcumin, when included in the culture medium throughout the experimental observation (72 hours), was able to significantly lower the increase of mAb C219-immunoreactive protein spontaneously occurring in the cells during culture. Curcumin, at doses ranging from 50 to 150microM was cytotoxic for freshly-plated hepatocytes, as shown by the strong decrease in the cell ability to exclude trypan blue 24 hours later, but it was significantly less cytotoxic when added to 24 or 48 hour-cultured cells. The resistance to curcumin, progressively acquired by cells during culture, was significantly reduced by high concentrations of dexamethasone (DEX) or dimethyl-sulfoxide (DMSO), culture conditions known to inhibit the spontaneous overexpression of Pgp. In addition, in a concentration-dependent manner, verapamil reverted curcumin resistance in Pgp overexpressing hepatocytes. In photoaffinity labeling studies, curcumin competed with azidopine for binding to Pgp, suggesting a direct interaction with glycoprotein. These results suggest that curcumin is able to modulate in vitro both expression and function of hepatic Pgp and support the hypothesis that curcumin, a chemopreventive phytochemical, could reveal itself also as a compound endowed with chemosensitizing properties on mdr phenotype.     

In vitro toxicity of zamifenacin (UK-76,654) and metabolites in primary hepatocyte cultures

1. We compared the sensitivities of primary hepatocytes from rat, dog and monkey to zamifenacin and two major metabolites, the methylenedioxy ring-opened catechol, UK-80,178 and its methylated product, UK-82,201. Toxicity was determined both via neutral red uptake and enzyme leakage data. 2. Canine hepatocytes were most sensitive to the cytotoxic effects of zamifenacin during 24-h exposure. Significant decreases in medium concentrations of zamifenacin in the presence of primary hepatocytes verified cellular uptake during the initial 2-h incubation. All three cell types were much more sensitive to UK-82,201 than to the catechol metabolite or parent drug. 3. The rapid onset of cytotoxicity indicated by elevations of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and other markers in the medium after UK-82,201 exposure, the delayed but substantial cytotoxic response to the parent drug which was suggestive of biotransformation to a reactive moiety, in vivo and in vitro drug metabolism results and subacute toxicology data suggest that dog may more effectively transform zamifenacin into UK-82,201, which is relatively hepatotoxic. 4. Because the catechol was generally less toxic than the O-methylated product, species that eliminate zamifenacin primarily as the catechol or its conjugate may be less affected by the potential hepatotoxicity of the methylated product. Our studies show that dog is the most sensitive species due to metabolism of the common catechol metabolite. The low incidence of potential hepatotoxicity in the clinic points to rare but important differences in the metabolism of Zamifencin. We conclude that the findings in dog were not predictive of subsequent effects in man.     

Differential induction of cytochrome P450 gene expression by 4n-alkyl-methylenedioxybenzenes in primary rat hepatocyte cultures

A well-characterized primary rat hepatocyte culture system was used to examine induction patterns of cytochrome 450 gene expression by a series of 4-n-alkyl-methylenedioxybenzene (MDBs) derivatives. Hepatocytes were treated for 24, 48, or 72 hours with 0-500 microM of the MDB compounds, and total cellular RNA and protein from each treatment was evaluated by hybridization and immunochemical techniques. Exposure to MDB congeners possessing increasing 4-n-alkyl side-chain length (C0-C8) resulted in dose- and structure-dependent activation of CYP2B1, 2B2, 3A1, 1A1, and 1A2 gene expression. At equivalent 100 microM concentrations, the C6 and C8 MDB congeners were more effective than the prototypical inducer phenobarbital (PB) with respect to induction potency of CYP2B1, CYP2B2, and CYP3A1 gene expression. In contrast to PB, longer side-chain-substituted MDBs effectively induced CYP1A1 and CYP1A2 gene expression, in addition to the CYP2B and CYP3A genes. At equivalent molar concentrations, the catechol derivative of C6-MDB was ineffective in its ability to induce CYP gene expression, indicating the importance of the intact methylenedioxy bridge in the induction mechanism. Levels of MDB-inducible CYP2B1 and CYP2B2 mRNA were highly correlated with CYP2B1/2 apoprotein levels, ascertained by immunoblot analysis of cultured hepatocyte S9 fractions. Compared with results from previous in vivo analysis (12), the current data indicate that pharmacodynamic factors may influence MDB induction profiles and that differences in MDB effects on CYP gene expression result depending on distinct structure-activity relationships.     

Macrophage-induced inhibition of nitric oxide production in primary rat hepatocyte cultures via prostaglandin E2 release

Kupffer cells and other macrophages play an important role in pathogenesis of toxicants in the liver. The aim of this study was to evaluate the effect of macrophages on hepatocyte production of nitric oxide (NO), which has been previously reported to be protective toward oxidative stress induced in primary rat hepatocytes. For this purpose, RAW 264.7 macrophages were added to primary rat hepatocytes at various ratios between macrophages and hepatocytes. These cocultures were supplemented with lipopolysaccharide (LPS) and interferon gamma (IFN-gamma) for 23 hours to induce NO synthase and trigger NO production. NO production was followed by quantification of nitrites in culture medium and dinitrosyl iron complexes (DNIC) in intact hepatocytes after separation from macrophages. In cocultured hepatocytes incubated with LPS and IFN-gamma, DNIC and nitrite levels decreased compared with those observed in hepatocytes cultured without macrophages in the same conditions. Moreover, inhibition of NO production in hepatocyte cocultures was macrophage-number-dependent. Macrophage-conditioned medium also inhibited NO production in hepatocytes, suggesting that the effect of macrophages was mediated by soluble factors. Among the soluble factors known to decrease NO levels are some cytokines, growth factors, reactive oxygen species, and prostaglandins. Ultrafiltration of macrophage-conditioned medium through a 500-d membrane to rule out higher-molecular-weight molecules, such as anti-inflammatory cytokines and growth factors, failed to restore NO production. In the same way, the use of superoxide dismutase (SOD) and catalase (CAT) to eliminate reactive oxygen species produced by macrophages did not lead to recovery of NO levels in hepatocytes. However, when NO synthesis was inhibited in macrophages by NG-monomethyl-L-arginine (L-NMMA), hepatocytes recovered the capacity to produce NO. A net decrease of prostaglandin E2 (PGE2) release by macrophages was concomitantly observed. Moreover, inhibition of PGE2 production in macrophages by indomethacin led to restoration of NO levels. Taken together, our observations suggest that NO synthesized by macrophages can decrease NO production in hepatocytes via PGE2 release. Because of the protective role of NO toward many liver injuries, it may be postulated that macrophages contribute through this mechanism to liver damage.     

Correlation of increased mortality with the suppression of radiation-inducible microsomal epoxide hydrolase and glutathione S-transferase gene expression by dexamethasone: effects on vitamin C and E-induced radioprotection

Previous studies in this laboratory have shown that gamma-ray ionizing radiation in combination with oltipraz, a radioprotective agent, enhances hepatic microsomal epoxide hydrolase (mEH) and glutathione S-transferase (GST) expression. The present study was designed to investigate the effects of dexamethasone on the radiation-inducible expression of mEH and rGST genes and on the vitamin C and E-induced radioprotective effects in association with the expression of the genes. Treatment of rats with a single dose of dexamethasone (0.01-1 mg/kg, p.o.) caused a dose-dependent decrease in the constitutive mEH gene expression at 24 hr. The radiation-inducible mEH mRNA level (threefold increase after 3 Gy gamma-irradiation) was decreased by 21% and 88% by dexamethasone at the doses of 0.1 and 1 mg/kg, respectively. Although dexamethasone alone caused 2- to 5-fold increases in the hepatic rGSTA2 mRNA level, rats treated with dexamethasone prior to 3 Gy irradiation exhibited 80%-93% suppression in the radiation-inducible increases in the rGSTA2 mRNA level. The inducible rGSTA3 and rGSTA5 mRNA levels were also significantly decreased by dexamethasone, whereas the rGSTM1 mRNA level was reduced to a lesser extent. Vitamin C and/or E, however, failed to enhance the radiation-inducible increases in hepatic mEH and rGST mRNA levels. Whereas rats exposed to 3 Gy irradiation with or without vitamin C treatment (30 or 200 mg/kg/day, p.o., 2 days) exhibited approximately threefold increases in the mEH and rGSTA2/3/5 mRNA levels relative to untreated animals, dexamethasone treatment (1 mg/kg, p.o.) resulted in 64%-96% decreases in the mRNA levels at 24 hr. The inducible rGSTM1/2 mRNA levels in the vitamin C/E-treated rats were approximately 50% suppressed by dexamethasone. Although vitamin C and/or E treatment (200 mg/kg/day, p.o., 2 days) improved the 30-day survival rates of the 8 Gy gamma-irradiated mice from 39% up to 74%, the improved survival rate of gamma-irradiated animals was reduced to 30% by dexamethasone pretreatment (1 mg/kg/day, 2 days). The mean survival time of dexamethasone-treated animals was reduced to approximately 2 days from 14 days in the animals with total body irradiation alone. No significant hematologic changes were observed in mice at 10 days after dexamethasone plus gamma-irradiation, as compared with irradiation alone. These results demonstrate that: dexamethasone substantially suppresses radiation-inducible mEH, rGSTA and rGSTM expression in the liver; vitamins C/E exhibit radioprotective effects without enhancing radiation-inducible mEH and GST gene expression; and inhibition of radiation-inducible mEH and rGST gene expression in the vitamin C- and E-treated animals by dexamethasone was highly correlated with reduction in the survival rate and the mean survival time of gamma-irradiated animals.     

Effects of propofol on human hepatic microsomal cytochrome P450 activities

1. The potential of propofol to inhibit the activity of major human cytochrome P450 enzymes has been examined in vitro using human liver microsomes. Propofol produced inhibition of CYP1A2 (phenacetin O-deethylation), CYP2C9 (tolbutamide 4'-hydroxylation), CYP2D6 (dextromethorphan O-demethylation) and CYP3A4 (testosterone 6beta-hydroxylation) activities with IC50 = 40, 49, 213 and 32 microM respectively. Ki for propofol against all of these enzymes with the exception of CYP2D6, where propofol showed little inhibitory activity, was 30, 30 and 19 microM respectively for CYPs 1A2, 2C9 and 3A4. 2. Furafylline, sulphaphenazole, quinidine and ketoconazole, known selective inhibitors of CYPs 1A2, 2C9, 2D6 and 3A4 respectively, were much more potent than propofol having IC50 = 0.8, 0.5, 0.2 and 0.1 microM; furafylline and sulphaphenazole yielded Ki = 0.6 and 0.7 microM respectively. 3. The therapeutic blood concentration of propofol (20 microM; 3-4 microg/ml) together with the in vitro Ki estimates for each of the major human P450 enzymes have been used to estimate the extent of cytochrome P450 inhibition, which may be produced in vivo by propofol. This in vitro-in vivo extrapolation indicates that the degree of inhibition of CYP1A2, 2C9 and 3A4 activity which could theoretically be produced in vivo by propofol is relatively low (40-51%); this is considered unlikely to have any pronounced clinical significance. 4. Although propofol has now been used in > 190 million people since its launch in 1986, there are only single reports of possible drug interactions between propofol and either alfentanil or warfarin. Consequently, it is difficult to conclude from either the published literature or the ZENECA safety database whether there is any evidence to indicate that propofol produces clinically significant drug interactions through inhibition of cytochrome P450-related drug metabolism.