Masakazu Isobe

Involvement of Carboxylesterase 1 and 2 in the Hydrolysis of Mycophenolate Mofetil

Mycophenolate mofetil (MMF) is the ester prodrug of the immunosuppressant agent mycophenolic acid (MPA) and is rapidly activated by esterases after oral administration. However, the role of isoenzymes in MMF hydrolysis remains unclear. Although human plasma, erythrocytes, and whole blood contain MMF hydrolytic activities, the mean half-lives of MMF in vitro were 15.1, 1.58, and 3.20 h, respectively. Thus, blood esterases seemed to contribute little to the rapid MMF disappearance in vivo. In vitro analyses showed that human intestinal microsomes exposed to 5 and 10 μM MMF exhibited hydrolytic activities of 2.38 and 4.62 nmol/(min · mg protein), respectively. Human liver microsomes exhibited hydrolytic activities of 14.0 and 26.1 nmol/(min · mg protein), respectively, approximately 6-fold higher than those observed for intestinal microsomes. MMF hydrolytic activities in human liver cytosols were 1.40 and 3.04 nmol/(min · mg protein), respectively. Because hepatic cytosols generally contain 5-fold more protein than microsomes, MMF hydrolysis in human liver cytosols corresponded to approximately 50% of that observed in microsomes. Fractions obtained by 9000g centrifugation of supernatants from COS-1 cells expressing human carboxylesterase (CES) 1 or 2 exhibited MMF hydrolytic activity, with CES1-containing fractions showing higher catalytic efficiency than CES2-containing fractions. The CES inhibitor bis-p-nitrophenylphosphate inhibited MMF hydrolysis in human liver microsomes and cytosols with IC50 values of 0.51 and 0.36 μM, respectively. In conclusion, both intestinal and hepatic CESs and in particular CES1 may be involved in MMF hydrolysis and play important roles in MMF bioactivation. Hepatic CES1 activity levels may help explain the between-subject variability observed for MMF usage.

Studies on the lipase of Chromobacterium viscosum: III. Purification of a low molecular weight lipase and its enzymatic properties

Abstract One of the lipases (glycerol ester hydrolase, EC 3.1.1.3) of Chromobacterium viscosum was purified from the original crude powder by approx. 1000-fold to give a yield of 18%. The purification method consisted of chromatography on Sephadex G-100, CM-cellulose and DEAE-Sephadex A-50. The homogeneity of the preparation was demonstrated by disc electrophoresis. A molecular weight of 27 000 was obtained by gel filtration and the isoelectric point in pI 6.9 was determined by isoelectric focusing. The optimum pH for the hydrolysis of olive oil was 6.5 and the optimum temperature was 70 °C in the standard assay system. The purified enzyme was stable in the range of pH 4–11 and below 40 °C in the absence of substrate. The lipase had a resistance for the inactivation by various metal ions and chemical reagents. Neither acceleration nor inhibition of the activity by bile salts was observed.

Cholesterol α- and β-epoxides as obligatory intermediates in the hepatic microsomal metabolism of cholesterol to cholestanetriol

Abstract A high performance liquid Chromatographie method for the good separation and direct determination of cholesterol a-epoxide (5,6α-epoxy-5α-cholestan-3β-ol) and β-epoxide (5,6β-epoxy-5β-cholestan-3β-ol) was introduced to the study of microsomal lipid peroxidation-mediated oxygenation of the cholesterol double bond. In the presence of NADPH, FeSO 4 , and ADP, bovine liver microsomes converted [4- 14 C] cholesterol to the α-epoxide, β-epoxide, and cholestanetriol (5α-cholestane-3β,5,6β-triol) in the ratio 1.0:4.3:0.7. Obligatory intermediacy of both cholesterol α- and β-epoxides and essential role of microsomal cholesterol epoxide hydratase in the conversion of cholesterol to cholestanetriol were established by using the isotope trapping method as well as the cholesterol epoxide hydratase inhibitor, 5,6α-imino-5α-cholestan-3β-ol. Hepatic microsomal P-450 played no appreciable role in the epoxidation of cholesterol. Microsomal cholesterol epoxide hydratase was with no doubt found to differ in nature from microsomal xenobiotic epoxide hydratase. Microsomal hydrolysis of styrene oxide and safrole oxide (0.1 mM each) was almost completely inhibited by 3,3,3-trichloro-1-propene oxide (1 mM) but not by 5,6α-imino-5α-cholestan-3β-ol (1 mM). However, microsomal hydrolysis of both cholesterol α- and β-epoxides was remarkably accelerated by 3,3,3-trichloro-1-propene oxide and inhibited by 5,6α-imino-5α-cholestan-3β-ol.

Differences in liver homogenates from Donryu, Fischer, Sprague-Dawley and Wistar strains of rat in the drug-metabolizing enzyme assay and the salmonella/hepatic S9 activation test

Comparison studies for detecting differences between liver microsome and S9 preparations from 4 strains (Donryu, Fischer, Sprague-Dawley, Wistar) of young male rats were carried out with pretreatment of the animals by inducers such as PCBs and PB plus 5,6-BF. Each microsome fraction was assayed for the enzymic activity of metabolism of model substrates such as aniline, benzophetamine, BP, DMN and 7-ethoxycoumarin. The hepatic S9 sample was also compared, as regards its metabolizing ability to activate 9 pre-mutagens (2AA, AAF, o-AAT, BP, DAB, DMBA, DMN, m-PDA, quinoline) to directly acting mutagens in the Salmonella/hepatic S9 activation test by using TA98, TA100 and TA1537 strains with or without cytochrome P450 inhibitors (SKF-525A, metyrapone, 7,8-benzo-flavone). In the enzymic assay with PCBs-induced microsomes, BP hydroxylation a strain-specific difference: the microsomes from Fischer and Wistar rats were more effective for metabolizing BP than those from the other strains of rat. The effect of induction by BP plus 5,6-BF for Fischer rats showed relatively higher enzymic activity in the same induction group. Other microsomes prepared from rats with and without induction by PB plus, 5,6-BF did not show a clear-cut strain dependency in the enzymic activities assayed. In the mutation experiments with hepatic S9 samples, the examination of DAB and quinoline revealed a marked strain difference when S9 samples prepared from PCBs-pretreated and PB-plus-5,6-BF-induced rats were used: the S9 sample from Fischer rats was available for activating the two pre-mutagens to directly acting mutagens. No marked difference in the metabolic activation of the remaining 7-pre-mutagens was observed on other S9 preparations. In examinations of mutagenicity activities with the use of three inhibitors, the two S9 preparations made with the two induction methods showed inhibition profiles closely similar to each other. However, there were minor differences in the profiles by these inhibitors. From these findings it was concluded that Fischer rat-liver S9 is useful for detecting mutagens in the metabolic activation test, when induction by PB plus 5,6-BF was used in the Ames Salmonella test.

Chromium(VI) inhibits mouse metallothionein-I gene transcription by preventing the zinc-dependent formation of an MTF-1-p300 complex.

Mouse MT-I (metallothionein-I) transcription is regulated by MTF-1 (metal-response-element-binding transcription factor-1) which is recruited to the promoter in response to zinc. Cr(VI) [chromium(VI)] pretreatment blocks zinc-activation of the endogenous MT-I gene and attenuates zinc-activation of MT-I-promoter-driven luciferase reporter genes in transient transfection assays. Chromatin immunoprecipitation assays revealed that Cr(VI) only modestly reduces recruitment of MTF-1 to the MT-I promoter in response to zinc, but drastically reduces the recruitment of RNA polymerase II. These results suggest that Cr(VI) inhibits the ability of MTF-1 to transactivate this gene in response to zinc. Zinc has recently been shown to induce the formation of a co-activator complex containing MTF-1 and the histone acetyltransferase p300 which plays an essential role in the activation of MT-I transcription. In the present study, co-immunoprecipitation assays demonstrated that Cr(VI) pretreatment blocks the zinc-induced formation of this co-activator complex. Thus Cr(VI) inhibits mouse MT-I gene expression in response to zinc by interfering with the ability of MTF-1 to form a co-activator complex containing p300 and recruiting RNA polymerase II to the promoter.

Ethanol-induced expression of glutamate–cysteine ligase catalytic subunit gene is mediated by NF-κB

Glutamate-cysteine ligase is a rate-limiting enzyme in the de novo synthesis of glutathione, a known scavenger of electrophiles and reactive oxygen species. Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by nuclear factor erythroid 2-related factor 2 (Nrf2). It has been reported that ethanol induces human GCLC production via Nrf2-mediated transactivation of the antioxidant-responsive element (ARE). Here, the luciferase reporter assay revealed the presence of an ethanol-responsive element in the human GCLC promoter; it spanned bases -1432 to -832 in hepatocytes and HepG2 cells transfected with cytochrome P450 2E1 (CYP2E1). The region lacked an ARE but had a putative nuclear factor-kappaB (NF-kappaB) element. NF-kappaB DNA-binding activity was activated in response to ethanol treatment. CYP2E1 expression was required for GCLC promoter-driven gene expression and the activation of NF-kappaB. Thus ethanol-induced GCLC transcription is mediated by not only Nrf2 but also NF-kappaB.

Cloning and sequence analysis of a hamster liver cDNA encoding a novel putative carboxylesterase

A full-length cDNA encoding for a putative carboxylesterase was isolated from a hamster liver cDNA library. The cDNA consisting of 1911 base pairs contained an open reading frame of 1683 base pairs encoding for a polypeptide of 561 amino-acid residues, including 27 N-terminal amino-acid residues for signal peptide. The deduced amino-acid sequence of the cDNA is in 67% homology with the amino-acid sequence of rabbit form 2 carboxylesterase, which has not yet been cloned. It also had many structural features highly conserved among carboxylesterase isozymes.

Studies on metabolism and toxicity of styrene: III. The effect of metabolic inactivation by rat-liver S9 on the mutagenicity of phenyloxirane toward Salmonella typhimurium

A comparative study on enzymic factors influencing the metabolic inactivation of phenyloxirane (styrene oxide), a major mutagenic metabolite of styrene in the liver, was carried out with respect to soluble glutathione S-transferase and microsomal epoxide hydratase in the 9000 X g supernatant fraction (S9) from a rat-liver homogenate. The mutagenic activity of phenyloxirane to Salmonella typhimurium TA100 was markedly reduced by S9 in the presence of glutathione but to a smaller extent in its absence. The retarding effect of glutathione on the inherent mutagenic activity of phenyloxirane was exerted by the soluble supernatant of S9 but not by microsomes. A gas-liquid chromatographic study indicated that the effect of glutathione was attributable to the disappearance of the mutagen from the microbial assay system. The rate of the disappearance was 10-20 times as fast in the soluble supernatant fraction as in the microsomes when fortified with more than 4 mM glutathione. Our results strongly suggest that in hepatic cells of the rat, cytosol glutathione S-transferase plays a much more important role than microsomal epoxide hydratase in the detoxication of the metabolite, phenyloxirane.

Epoxidation of cholesterol by hepatic microsomal lipid hydroperoxides

[1,2-3H]Cholesterol was epoxidized to radioactive cholesterol α- and β-epoxides (5,6α-epoxy-5α- and 5,6β-epoxy-5β-cholestan-3β-ols) in the ratio 1:4 by hepatic microsomal lipid hydroperoxides (MsOOH, 1 mM as active oxygen) in the presence of ferrous ion. MsOOH could be replaced by methyl linoleate hydroperoxides (MOOH) under the same conditions although the latter was less effective than the former. None of cumene hydroperoxide, t-butyl hydroperoxide, and hydrogen peroxide was an effective oxidant even at 10 mM. Neither ADP nor EDTA had an effect on the epoxidation of cholesterol by MsOOH as well as by MOOH. Ferrous ion could not be replaced by ferric ion in the hydroperoxide-mediated epoxidation. Cyanide anion potentially inhibited the reaction.

The zinc-sensing transcription factor MTF-1 mediates zinc-induced epigenetic changes in chromatin of the mouse metallothionein-I promoter.

Metallothionein (MT) is a small, cysteine-rich protein active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Mouse MT-I gene transcription is regulated by metal response element-binding transcription factor-1 (MTF-1), which is recruited to the promoter by zinc. We examined alterations in the chromatin structure of the MT-I promoter associated with enhanced transcriptional activation. MTF-1 proved essential for zinc-induced epigenetic changes in the MT-I promoter. Chromatin immunoprecipitation assays demonstrated that zinc treatment rapidly decreased Lys⁴-trimethylated and Lys⁹-acetylated histone H3 in the promoter and decreased total histone H3 but not histone H3.3. Micrococcal nuclease sensitivity of the MT-I promoter was increased by zinc. Thus, the chromatin structure in the promoter may be locally disrupted by zinc-induced nucleosome removal. Without MTF-1 these changes were not observed, and an MTF-1 deletion mutant recruited to the MT-I promoter by zinc that did not recruit the coactivator p300 or activate MT-I transcription did not affect histone H3 in the MT-I promoter in response to zinc. Interleukin-6, which induces MT-I transcription independently of MTF-1, did not reduce histone H3 levels in the promoter. Rapid disruption of nucleosome structure at the MT-I promoter is mediated by zinc-responsive recruitment of an active MTF-1-coactivator complex.