Sheep Liver Microsomes Research Articles

Characterisation of cytochrome P450 isoenzyme activity in sheep liver and placental microsomes

IntroductionDrug metabolism during pregnancy is a complex process that involves maternal, placental and fetal sites of metabolism. Indeed, there is a lack of clarity provided from drug metabolism in human pregnancy due to ethical limitations. Large animal models of human pregnancy provide an opportunity to quantify activity of phase 1 drug metabolism mediated by cytochrome P450 (CYP) enzymes in the maternal, placental, and fetal compartments. Herein, we have validated a comprehensive assay to quantify maternal, placental, and fetal CYP activity. MethodsIsolated microsomes from sheep maternal liver, placenta, and fetal liver (140d gestation, term = 150d) were incubated with CYP-specific probe drugs to quantify the activity of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A. Inhibition studies were performed to validate specificity of probe drugs. The validated assay was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). ResultsCYP1A2, CYP2B6, CYP2C8, CYP2C19, CYP2D6, CYP2E1 and CYP3A were active in maternal liver. In contrast, only CYP1A2, CYP2C8 and CYP2D6 were active in the placenta, whereas CYP2B6, CYP2C8 and CYP2D6 were active in the fetal liver. Of the placental-specific CYPs validated, CYP1A2 increased in type A compared with type D placentomes, whereas CYP2C8 activity increased in type B compared with type A and C. DiscussionThis study has established conditions for compartment-specific CYP activity in the sheep maternal-placental-fetal unit using a validated and standardised experimental workflow. Compartment- and placentome type-specific CYP activity are important considerations when examining drug metabolism in the maternal-placental-fetal unit and in determining the impact of pregnancy complications.

Pharmacological characterization of geraniol in sheep and its potential use in the control of gastrointestinal nematodes

Geraniol (GNL) was effective against gastrointestinal nematodes in vitro; nevertheless, the anthelmintic effect of phytochemicals combined with synthetic drugs has been little explored in vivo. This article characterized in vitro / in vivo the pharmacological features of GNL in sheep as well as its pharmacokinetic interaction with albendazole (ABZ). Additionally, the in vivo efficacy of GNL against Haemonchus contortus was evaluated in lambs. Liver microsomes from lambs were incubated in the absence or presence of GNL to analyze CYP1A1, CYP1A2 and FMO metabolic pathways. The effect of GNL on the hepatic sulfoxidation and sulfonation of ABZ and the ruminal sulforeduction of albendazole sulfoxide (ABZSO) was assessed. The in vivo pharmacokinetic interaction of ABZ and GNL was evaluated in lambs. The effect of GNL on the fecal egg count was evaluated in lambs infected with a resistant isolate of H. contortus. In sheep liver microsomes, the presence of 2 mM GNL reduced the CYP1A1, CYP1A2 and FMO pathways by 77.9, 90.8 and 84.5%, respectively, with respect to control (P < 0.05). In the presence of 2 mM GNL, the ABZ sulfoxidation decreased from 114.4 ± 8.49 (control) to 50.24 ± 11.1 nmol/min.mg, and ABZSO2 production decrease from 0.52 ± 0.14 to 0.09 ± 0.03 nmol/h.mg. No changes in the pharmacokinetic behavior of ABZ were observed in the presence of GNL. The in vivo efficacy of four doses of GNL was 40.5%. These findings highlight the importance of integrated in vitro / in vivo pharmaco-parasitological studies to develop new pharmacological tools for controlling gastrointestinal parasites.

Tissue and interspecies comparison of catechol-O-methyltransferase mediated catalysis of 6-O-methylation of esculetin to scopoletin and its inhibition by entacapone and tolcapone

Catechol-O-methyltransferase (COMT) methylates both endogenous and exogenous catechol compounds to inactive and safe metabolites. We first optimised conditions for a convenient and sensitive continuous fluorescence-based 6-O-methylation assay of esculetin, which we used for investigating the COMT activity in human, mouse, rat, dog, rabbit, and sheep liver cytosols and microsomes and in ten different rat tissues. Furthermore, we compared the inhibition potencies and mechanisms of two clinically used COMT inhibitors, entacapone and tolcapone, in these species. In most tissues, the COMT activity was at least three times higher in cytosol than in microsomes. In the rat, the highest COMT activity was found in the liver, followed by kidney, ileum, thymus, spleen, lung, pancreas, heart, brain, and finally, skeletal muscle. Entacapone and tolcapone were characterised as highly potent mixed type tight-binding inhibitors. The competitive inhibition type dominated over the uncompetitive inhibition with entacapone, whereas uncompetitive inhibition dominated with tolcapone. Rats, dogs, pigs, and sheep are high COMT activity species, in contrast to humans, mice, and rabbits; COMT activity is highest in the liver. Both entacapone and tolcapone are potent COMT inhibitors, but their inhibition mechanisms differ.

Hepatic biotransformation pathways and ruminal metabolic stability of the novel anthelmintic monepantel in sheep and cattle.

Monepantel (MNP) is a new amino-acetonitrile derivative anthelmintic drug used for the treatment of gastrointestinal (GI) nematodes in sheep. The present work investigated the main enzymatic pathways involved in the hepatic biotransformation of MNP in sheep and cattle. The metabolic stability in ruminal fluid of both the parent drug and its main metabolite (monepantel sulphone, MNPSO2 ) was characterized as well. Additionally, the relative distribution of both anthelmintic molecules between the fluid and particulate phases of the ruminal content was studied. Liver microsomal fractions from six (6) rams and five (5) steers were incubated with a 40μm of MNP. Heat pretreatment (50°C for 2min) of liver microsomes was performed for inactivation of the flavin-monooxygenase (FMO) system. Additionally, MNP was incubated in the presence of 4, 40, and 80μm of methimazole (MTZ), a FMO inhibitor, or equimolar concentrations of piperonyl butoxide (PBx), a well-known general cytochrome P450 (CYP) inhibitor. In both ruminant species, MNPSO2 was the main metabolite detected after MNP incubation with liver microsomes. The conversion rate of MNP into MNPSO2 was fivefold higher (P<0.05) in sheep (0.15±0.08nmol/min·mg) compared to cattle. In sheep, the relative involvement of both FMO and CYP systems (FMO/CYP) was 36/64. Virtually, only the CYP system appeared to be involved in the production of MNPSO2 in cattle liver. Methimazole significantly reduced (41 to 79%) the rate of MNPSO2 production in sheep liver microsomes whereas it did not inhibit MNP oxidation in cattle liver microsomes. On the other hand, PBx inhibited the production of MNPSO2 in liver microsomes of both sheep (58 to 98%, in a dose-dependent manner) and cattle (almost 100%, independently of the PBx concentration added). The incubation of MNP and MNPSO2 with ruminal contents of both species showed a high chemical stability without evident metabolism and/or degradation as well as an extensive degree of adsorption (83% to 90%) to the solid phase of the ruminal content. Overall, these results are a further contribution to the understanding of the metabolic fate of this anthelmintic drug in ruminants.

Species-Specific Differences in the in Vitro Metabolism of Lasiocarpine.

There are species-related differences in the toxicity of pyrrolizidine alkaloids (PAs) partly attributable to the hepatic metabolism of these alkaloids. In this study, the metabolism of lasiocarpine, a potent hepatotoxic and carcinogenic food contaminant, was examined in vitro with human, pig, rat, mouse, rabbit, and sheep liver microsomes. A total of 12 metabolites (M1-M12) were detected with the human liver microsomes, of which M1, M2, M4, and M6 were unstable in the presence of reduced glutathione (GSH). With the exception of M3 and M8, the formation of all metabolites of lasiocarpine was catalyzed by CYP3A4 in humans. Tandem mass spectra (MS/MS) detected several new metabolites, termed M4-M7; their toxicological significance is unknown. M9 (m/z 398), identified as a demethylation product, was the main metabolite in all species, although the relative dominance of this metabolite was lower in humans. The level of the reactive metabolites, as measured by M1 ((3H-pyrrolizin-7-yl)methanol) and the GSH conjugate, was higher with the liver microsomes of susceptible species (human, pig, rat, and mouse) than with the species (rabbit and sheep) resistant to PA intoxication. In general, in addition to the new metabolites (M4-M7) that could make humans more susceptible to lasiocarpine-induced toxicity, the overall metabolite fingerprint detected with the human liver microsomes differed from that of all other species, yielding high levels of GSH-reactive metabolites.

In vitro and in vivo assessment of the benzydamine-mediated interference with the hepatic S-oxidation of the anthelmintic albendazole in sheep

The aim of this research was to investigate the influence of benzydamine (BZ) on the in vitro and in vivo hepatic metabolism of the anthelmintic albendazole (ABZ) in sheep. The enantioselective ABZ S-oxidation was assessed by the amount of its (−) and (+) ABZ-sulphoxide (ABZSO) enantiomers formed in sheep liver microsomes (in vitro work). In the in vivo trial, lambs received ABZ (5mg/kg, intra-ruminal route) or ABZ (5mg/kg) plus BZ (8mg/kg, i.m., two doses 4h apart). Incubated and plasma samples were analysed by HPLC. In vitro, BZ IC50s (the concentrations that produced a 50% decrease in ABZ S-oxidation) for the production of total ABZSO and (+)ABZSO were 71.0±8.1 and 62.6±8.1μM, respectively. BZ showed a strong inhibitory potency over the flavin-monooxygenase (FMO)-dependent production of (+)ABZSO compared to the cytochrome P450 (CYP)-mediated production of (−)ABZSO. In vivo, co-administration of BZ with ABZ did not change the pharmacokinetic parameters of ABZSO and ABZSO2 with the exception of significantly higher (p<0.01) formation half-lives (t1/2for) for (−)ABZSO (3.24±1.03h vs. 6.19±2.18h) and (+)ABZSO (3.87±1.20h vs. 7.21±2.46h). BZ inhibited the hepatic FMO and CYP-dependent S-oxidation of ABZ in vitro. However, the metabolic interaction between ABZ and BZ was not observed in the in vivo pharmacokinetic trial. Hence, further work using a different dosing scheme or pharmaco-technical preparation of BZ may be required to observe in vivo the metabolic interference clearly shown under in vitro conditions.

In vitro effects of pesticide exposure on the activity of the paraoxonase-1 enzyme from sheep liver microsomes

The organophosphate target enzyme paraoxonase-1 (PON1) has been extensively studied in toxicology. Pesticides are organophosphate compounds that are commonly used in agriculture. They are also used as nerve gases, such as sarin and soman. Therefore, the effects of these compounds on enzymatic activity are well known. In the present study, we investigated the in vitro effects of fenoxaprop-p-ethyl, lambda cyhalothrin, imidacloprid, and dichlorvos pesticides on sheep liver microsomal PON1. The enzyme was purified approximately 141-fold with a specific activity of 1822.22 EU/mg proteins. The pesticides inhibited sheep liver PON1 in vitro, and the IC50 values for these compounds were 0.0103, 0.069, 0.157, and 0.2 m M, respectively.

Hepatic and extra-hepatic metabolic pathways involved in flubendazole biotransformation in sheep

Flubendazole (FLBZ) is a broad-spectrum benzimidazole anthelmintic compound used in pigs, poultry and humans. Its potential for parasite control in ruminant species is under investigation. The objective of the work described here was to identify the main enzymatic pathways involved in the hepatic and extra-hepatic biotransformation of FLBZ in sheep. Microsomal and cytosolic fractions obtained from sheep liver and duodenal mucosa metabolised FLBZ into a reduced FLBZ metabolite (red-FLBZ). The keto-reduction of FLBZ led to the prevalent (∼98%) stereospecific formation of one enantiomeric form of red-FLBZ. The amounts of red-FLBZ formed in liver subcellular fractions were 3–4-fold higher ( P < 0.05) compared to those observed in duodenal subcellular fractions. This observation correlates with the higher ( P < 0.05) carbonyl reductase (CBR) activities measured in the liver compared to the duodenal mucosa. No metabolic conversion was observed following FLBZ or red-FLBZ incubation with sheep ruminal fluid. Sheep liver microsomes failed to convert red-FLBZ into FLBZ. However, this metabolic reaction occurred in liver microsomes prepared from phenobarbital-induced rats, which may indicate a cytochrome P450-mediated oxidation of red-FLBZ. A NADPH-dependent CBR is proposed as the main enzymatic system involved in the keto-reduction of FLBZ in sheep. CBR substrates such as menadione and mebendazole (a non-fluoride analogue of FLBZ), inhibited this liver microsomal enzymatic reaction, which may confirm the involvement of a CBR enzyme in FLBZ metabolism in sheep. This research is a further contribution to the understanding of the metabolic fate of a promissory alternative compound for antiparasitic control in ruminant species.

Kinetics of Valproic Acid Glucuronidation: Evidence for in Vivo Autoactivation

Sigmoidal or autoactivation kinetics has been observed in vitro for both cytochrome P450- and UDP-glucuronosyltransferase-catalyzed enzymatic reactions. However, the in vivo relevance of sigmoidal kinetics has never been clearly demonstrated. In the current study we investigate the kinetics of valproic acid glucuronide (VPAG) formation both in vivo in adult sheep and in vitro in sheep liver microsomes (pool of 10). After a 100 mg/kg i.v. bolus dose of valproic acid (VPA) to adult sheep (n = 5), the majority of the dose was recovered in urine as VPAG (approximately 79%). Eadie-Hofstee plots of the VPAG formation rate (calculated from urinary excretion rate data for VPAG) were characteristic of autoactivation kinetics and provided estimates of the apparent maximum velocity of an enzymatic reaction (V(max)(app)), the substrate concentration resulting in 50% of V(max)(app) (S(50)(app)), and Hill coefficient (n) of 2.10 +/- 0.75 micromol/min/kg, 117 +/- 56 microM, and 1.34 +/- 0.14, respectively. Comparable estimates of V(max)(app) (2.63 +/- 0.33 micromol/min/kg), S(50)(app) (118 +/- 53 microM), and n (2.06 +/- 0.47) describing overall VPA elimination from plasma were obtained by fitting VPA unbound plasma concentration-time data to a two-compartment model with elimination described by the Hill equation. Consistent with our in vivo observations, Eadie-Hofstee plots of VPAG formation in sheep liver microsomes were characteristic of autoactivation kinetics. To our knowledge, these data provide the first clear demonstration that autoactivation kinetics observed in vitro in liver preparations can translate to the in vivo situation at least under certain experimental conditions and confirm its relevance.

Assessment of the main metabolism pathways for the flukicidal compound triclabendazole in sheep

Triclabendazole (TCBZ) is an halogenated benzimidazole (BZD) compound worldwide used to control immature and adult stages of the liver fluke Fasciola hepatica. The purpose of this investigation was to characterize in vitro the patterns of hepatic and ruminal biotransformation of TCBZ and its metabolites in sheep. TCBZ parent drug was metabolized into its sulphoxide (TCBZSO), sulphone (TCBZSO2) and hydroxy derivatives by sheep liver microsomes. The same microsomal fraction was also able to oxidize TCBZSO into TCBZSO2 and hydroxy-TCBZSO (HO-TCBZSO). TCBZ sulphoxidation was significantly (P < 0.001) inhibited after inactivation of the flavin-monooxygenase (FMO) system (77% inhibition) as well as in the presence of the FMO substrate methimazole (MTZ) (71% inhibition). TCBZ sulphoxidative metabolism was also reduced (24% inhibition, P < 0.05) by the cytochrome P450 inhibitor piperonyl butoxide (PB). The rate of TCBZSO conversion into TCBZSO2 was also significantly inhibited by PB (55% inhibition), MTZ (52% inhibition) and also following FMO inactivation (58% inhibition). The data reported here indicate that the FMO is the main enzymatic pathway involved in TCBZ sulphoxidation (ratio FMO/P450 = 3.83 +/- 1.63), although both enzymatic systems participate in a similar proportion in the sulphonation of TCBZSO to form the sulphone metabolite (ratio FMO/P450 = 1.31 +/- 0.23). Additionally, ketoconazole (KTZ) did not affect TCBZ sulphoxidation but decreased (66% inhibition, P < 0.05) the formation of TCBZSO2. Similarly, inhibition of TCBZSO2 production was observed after incubation of TCBZSO in the presence of KTZ and erythromycin (ETM). Conversely, thiabendazole (TBZ) and fenbendazole (FBZ) did not affect the oxidative metabolism of both incubated substrates. The sheep ruminal microflora was able to reduce the sulphoxide (TCBZSO) into the parent thioether (TCBZ). The ruminal sulphoreduction of the HO-TCBZSO derivative into HO-TCBZ was also demonstrated. The rate of sulphoreduction of HO-TCBZSO was significantly (P < 0.05) higher than that observed for TCBZSO. The metabolic approach tested here contributes to the identification of the different pathways involved in drug biotransformation in ruminant species. These findings on the pattern of hepatic and ruminal biotransformation of TCBZ and its main metabolites are a further contribution to the understanding of the pharmacological properties of widely used anthelmintics in ruminants. Comprehension of TCBZ metabolism is critical to optimize its flukicidal activity.

Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity.

The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 7). The presence of 0.1% Triton X-100 in the reaction mixture caused an increase of specific sheep liver microsomal FMO activity towards methimazole to 6.1 +/- 1.4 nmol methimazole oxidized min(-1) mg(-1) microsomal protein (mean +/- SE, n = 6). Metabolism of imipramine and chlorpromazine was measured by following the oxidation of cofactor NADPH spectrophotometrically at 340 nm. Sheep liver microsomal FMO activity towards imipramine and chlorpromazine was found to be 10.7 and 12.3 nmol NADPH oxidized min(-1) mg(-1) microsomal protein, respectively. Characterization of sheep liver enzyme was carried out using methimazole as substrate and the maximum FMO enzyme activity was detected at 37 degrees C and at pH 8.0. The apparent K(m) value of sheep liver microsomal FMO for methimazole was 0.118 mM. Effects of the detergents Triton X-100, Cholate, and Emulgen 913, on FMO activity were determined and FMO activity was found to increase with the addition of detergents to the reaction medium. Sheep liver microsomal FMO-catalysed methimazole oxidation was inhibited by imipramine and chlorpromazine when these drugs were used at high concentrations. Western blot-immunochemical analysis revealed the presence of FMO3 in sheep liver microsomes.

Integrated pharmacological assessment of flubendazole potential for use in sheep: disposition kinetics, liver metabolism and parasite diffusion ability1

Flubendazole (FLBZ) is a broad spectrum benzimidazole methylcarbamate anthelmintic widely used in poultry and swine. However, there is no information available on the pharmacological behaviour of FLBZ in ruminants. The work reported here was addressed to evaluate the potential of FLBZ for use in sheep. The integrated assessment included evaluation of FLBZ and metabolites plasma disposition kinetics, liver metabolism and ex vivo ability to diffuse into the cestode parasite Moniezia benedeni. In a cross-over kinetic study, six healthy Corriedale sheep were treated with FLBZ by intravenous (i.v.) (4% solution) and intraruminal (i.r.) (4% suspension) administrations at the same dosage (5 mg/kg) with a 21-day washout period between treatments. Blood samples were collected between 0 and 72 h post-treatments. Sheep liver microsomes were incubated with 40 microm FLBZ and specimens of the cestode parasite M. benedeni, collected from untreated animals, were incubated (5-120 min) with FLBZ and its reduced (R-FLBZ) metabolite (5 microm). Samples of plasma, microsomal incubations and parasite material were prepared and analyzed by high-performance liquid chromatography to measure FLBZ and its metabolites. FLBZ parent drug showed a fast disposition being detected in the bloodstream up to 36 h after its i.v. administration. Both R-FLBZ and hydrolyzed FLBZ (H-FLBZ) metabolites were recovered in plasma as early as 5 min after the i.v. treatment in sheep. The plasma AUC ratios for R-FLBZ and FLBZ (AUC(R-FLBZ)/AUC(FLBZ)) were 4.07 i.v. and 5.55 i.r., respectively. R-FLBZ achieved a significantly higher (P < 0.01) C(max) value (0.14 microg/mL at 17.3 h post-treatment) than that observed for the parent drug FLBZ (0.04 microg/mL at 14.4 h post-treatment). Low plasma concentrations of FLBZ parent drug were measured between 6 and 48 h, and only trace concentrations of H-FLBZ were detected during a short period of time after the i.r. treatment. Consistently, sheep liver microsomes metabolized FLBZ into its reduced metabolite at a rate of 9.46 +/- 2.72 nmol/mg/h. Both FLBZ and R-FLBZ demonstrated a similar ability to quickly diffuse through the tegument of the cestode parasite. The data on FLBZ pharmacological behaviour presented here contribute to evaluate its potential to be developed as an anthelmintic for broad spectrum parasite control in ruminants.

Triclabendazole biotransformation and comparative diffusion of the parent drug and its oxidized metabolites into Fasciola hepatica

1. Triclabendazole (TCBZ) is an halogenated trematodicidal benzimidazole compound extensively used in veterinary medicine. It is active against immature and adult stages of the liver fluke Fasciola hepatica.2. Free and conjugated TCBZ metabolites have been identified in the bile of treated sheep.3. The experimental aims were to characterize the in vitro patterns of TCBZ biotransformation both in the animal host (sheep liver microsomes) and target parasite (F. hepatica microsomal preparation); and to compare the ex vivo diffusion of TCBZ parent drug and its oxidized metabolites (TCBZ sulphoxide [TCBZSO], TCBZ sulphone [TCBZSO2], and TCBZ-hydroxy derivatives) into F. hepatica. Additionally, the octanol–water partition coefficients for TCBZ and all its metabolites were estimated as an indicator of the relationship between drug lipophilicity and diffusion into the target parasite.4. Drug/metabolites concentrations were quantified by HPLC after sample clean up and a solvent-mediated chemical extraction.5. Sheep liver microsomes metabolized TCBZ into its sulphoxide and sulphone metabolites after 30 min of incubation. The rate of TCBZ sulphoxidation in the liver was significantly greater (p<0.01) than that observed for the sulphonation of TCBZSO.6. The trematode parasite oxidized TCBZ into its sulphoxide metabolite after 60 min of incubation at a metabolic rate of 0.09 nmol min−1 mg protein−1.7. TCBZ and all its oxidized metabolic products were recovered from F. hepatica as early as 15 min after their ex vivo incubation in a Kreb's Ringer Tris buffer. However, the diffusion of the hydroxy-derivatives into the fluke was lower than that observed for TCBZ, TCBZSO and TCBZSO2. There was a high correlation (r = 0.82) between drug lipophilicity (expressed as octanol–water partition coefficients) and drug availability measured within the parasite.8. Unlike the uptake pattern previously observed for albendazole, the parent TCBZ and its sulphoxide and sulphone metabolites showed a similar ability to penetrate into the trematode parasite.9. Understanding the relationship between TCBZ metabolism, the relative pharmacological potency of its metabolic products and their ability to reach the target parasite may be critical to optimize its flukicidal activity, particularly when TCBZ resistant flukes have been already isolated in the field.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization

A 25 kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na(2)CO(3), indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73-83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3 Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243-248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87-101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H(2)O(2). Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

Microsomal glutathione S-transferase A1-1 with glutathione peroxidase activity from sheep liver: molecular cloning, expression and characterization

A 25kDa subunit of glutathione S-transferase (GST) from sheep liver microsomes (microsomal GSTA1-1) with a significant selenium-independent glutathione peroxidase activity has been isolated and characterized. Several analytical criteria, including EDTA stripping, protease protection assay and extraction with alkaline Na2CO3, indicate that the microsomal GSTA1-1 is associated with the inner microsomal membrane. The specific cDNA nucleotide sequence reveals that the enzyme is made up of 222 amino acid residues and shares approx. 73–83% sequence similarity to Alpha-class GSTs from different species. The molecular mass, as determined by electrospray mass ionization, is 25611.3Da. The enzyme is distinct from the previously reported rat liver microsomal GST in both amino acid sequence and catalytic properties [Morgenstern, Guthenberg and DePierre (1982) Eur. J. Biochem. 128, 243–248]. The microsomal GSTA1-1 differs from the sheep liver cytosolic GSTs, reported previously from this laboratory, in its substrate specificity profile and molecular mass [Reddy, Burgess, Gong, Massaro and Tu (1983) Arch. Biochem. Biophys. 224, 87–101]. In addition to catalysing the conjugation of 4-hydroxynonenal with GSH, the enzyme also exhibits significant glutathione peroxidase activity towards physiologically relevant fatty acid hydroperoxides, such as linoleic and arachidonic acid hydroperoxides, as well as phosphatidylcholine hydroperoxide, but not with H2O2. Thus the microsomal GSTA1-1 isoenzyme might have an important role in the protection of biological membranes against oxidative damage.

Pirfenidone inhibits NADPH-dependent microsomal lipid peroxidation and scavenges hydroxyl radicals.

Pirfenidone (Pf), a new broad-spectrum anti-fibrotic agent, is known to offer protection against lung fibrosis in vivo in laboratory animals, and against mitogenesis and collagen formation by human lung fibroblasts in vitro. Because reactive oxygen species are thought to be involved in these events, we investigated the mechanism(s) by which Pf ameliorates oxidative stress and its effects on NADPH-dependent lipid peroxidation. Pf has been shown to cause inhibit NADPH-dependent lipid peroxidation in sheep liver microsomes in a dose-dependent manner. The concentration of Pf required to cause 50% inhibition of lipid peroxidation was approximately 6 mM. Pf was found to be ineffective as a superoxide radical scavenger. Pf was also ineffective in decomposing H2O2 and chelating iron. In deoxyribose degradation assays, Pf was a potent scavenger of hydroxyl radicals with a rate constant of 5.4 x 10(9) M(-1) sec(-1). EPR spectroscopy in combination with spin trapping techniques, using a Fenton type reaction and DMPO as a spin-trapping agent, Pf scavenged hydroxyl radicals in a dose-dependent manner. The concentration of Pf required to inhibit 50% signal height was approximately 2.5 mM. Because iron was used in the Fenton reaction, the ability of Pf in chelating iron was verified in a fluorescent competitive assay using calcein as the fluorescent probe. Pf up to 10 mM concentration was ineffective in chelating either Fe2+ or Fe3+ in this system. We propose that Pf exerts its beneficial effects, at least in part, through its ability to scavenge toxic hydroxyl radicals.

Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver

Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200±500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the purified sheep liver cytochrome b5 to transfer electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. The K m and V max values were calculated to be 0.088 μM cytochrome b5 and 315.8 μM cytochrome c reduced/min/mg enzyme, respectively. Also the reduction of cytochrome b5 by reductase was studied and K m and V max values were determined to be 5 μM cytochrome b5 and 5200 nmol cytochrome b5 reduced/min/mg enzyme, respectively. The K m and V max values for the cofactor NADH in the presence of saturating concentration of cytochrome b5 were found to be 0.0017 mM NADH and 6944 nmol cytochrome b5 reduced/min/mg enzyme, respectively. NADH-cytochrome b5 reductase was also partially purified from the same source, detergent solubilized sheep liver microsomes, by using two successive DEAE-cellulose, and 5′-ADP-agarose affinity column chromatographies. It was purified 144-fold and the yield was 7% with respect to microsomes. The apparent monomer Mr of reductase was estimated to be 34,000 by SDS-PAGE. When ferricyanide was used as an electron acceptor, reductase showed maximum activity between 6.8 and 7.5. The K m and V max values of the enzyme for ferricyanide were calculated as 0.024 mM ferricyanide and 673 μmol ferricyanide reduced/min/mg enzyme, respectively. The K m and V max values for the cofactor NADH in the presence of saturating amounts of ferricyanide were found to be 0.020 mM NADH and 699 μmol ferricyanide reduced/min/mg enzyme, respectively.

The Roles of CYP3A and CYP2B Isoforms in Hepatic Bioactivation and Detoxification of the Pyrrolizidine Alkaloid Senecionine in Sheep and Hamsters

The roles of cytochrome CYP3A and CYP2B isozymes in the bioactivation and detoxification of the pyrrolizidine alkaloid (PA) senecionine (SN) have been investigatedin vitrowith sheep and hamster hepatic microsomes. Our results show that the rate of SN activation measured by (±)-6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP) formation greatly exceeded the rate of SNN-oxide formation (detoxification) in hamsters. In contrast, SNN-oxide, a detoxification product, was the major metabolite in sheep with much lower DHP production. Immunoinhibition studies with anti-sheep CYP3A and CYP2B antibodies show that members of CYP3A subfamily play the major role in the conversion of PA to pyrrolic metabolites in both species (over 90% in sheep; 68% in hamster). These enzymes also contribute 38.8 and 41.3% of SN N-oxidation in sheep and hamsters, respectively. In contrast, CYP2B isoforms have a limited capacity toward DHP formation in both species (47% in sheep; 32% in hamster), while these enzymes catalyzed only 24.6 and 35.4% SN N-oxidation in sheep and hamster, respectively. Using triacetyloleandomycin (TAO) and gestodene, two highly selective chemical inhibitors of CYP3A isoforms, our data show that 90% of DHP formation was inhibited by either inhibitor in sheep. Gestodene appeared to be more efficient than TAO in the inhibition of DHP production in hamsters. Testosterone 6β-hydroxylase activity, a functional marker of CYP3A, was significantly inhibited by TAO and gestodene in sheep liver microsomes and by gestodene (100 μM) in hamster liver microsomes. These results suggest that CYP3A isozymes have important roles in bioactivation and detoxification of PA in both species, whereas CYP2B subfamily members are less efficient in biotransformation of PA.

Activity of N-acetylglucosamine-1-phosphate transferase in sheep liver microsomes: In vivo and in vitro inhibition by tunicamycin

The neurological disease of livestock known as annual ryegrass toxicity, caused by ingestion of bacterial toxins called corynetoxins, has been shown to be produced experimentally by injection of tunicamycin, a related antibiotic. In this study the effects of tunicamycin inhibition on the activity of the enzyme, N-acetylglucosamine-l-phosphate transferase, in sheep liver rough microsomes were measured in vitro and in vivo. Enzyme activity was dependent on Triton X-100 and exogenous dolichyl phosphate for maximal activity, although there was measurable activity in their absence. The transferase enzyme was very sensitive to in vitro (inhibition can be detected below 10 ng ml −1). In vivo, sheep treated parenterally with a single dose of tunicamycin showed a time and dose-dependent decrease in enzyme activity, which was almost completely inhibited for up to 14 days after a sublethal dose of toxin. In addition, the yield of rough microsomes was lower from toxin-treated sheep than from control animals.

Metyrapone is a competitive inhibitor of 11beta-hydroxysteroid dehydrogenase type 1 reductase.

The present study was designed to examine the effects of metyrapone in vitro on the activities of 11beta-hydroxysteroid dehydrogenase (11beta-HSD) types 1 and 2, the two intracellular enzymes responsible for the metabolism of glucocorticoids. Enzymatic activities of 11beta-HSD1 and 2 were determined by a radiometric conversion assay using cortisol and cortisone as physiological substrates. The enzyme activity assays were carried out in the absence and presence of metyrapone using sheep liver and kidney microsomes as the source of 11beta-HSD1 and 2, respectively. It was found that metyrapone inhibited the reductase activity of 11beta-HSD1 in a dose-dependent manner with an apparent Ki of 30 microM. Moreover, this inhibition was competitive because the Km for cortisone was increased in the presence of metyrapone. In contrast, metyrapone showed biphasic effects on the dehydrogenase activity of 11beta-HSD1, in that it increased the activity at concentrations lower than 100 microM but decreased it at higher concentrations. However, under similar conditions, metyrapone had little effect on the unidirectional dehydrogenase activity of 11beta-HSD2. In conclusion, the present results provide the first direct evidence that metyrapone is a competitive inhibitor of 11beta-HSD1 reductase, and that it also exerts biphasic effects on 11beta-HSD1 dehydrogenase activity. These findings indicate that metyrapone influences peripheral glucocorticoid metabolism through its regulation of 11beta-HSD1 activity, in addition to its classic inhibitory effects on adrenal steroid biosynthesis. It is therefore imperative that this novel extra-adrenal effect of metyrapone be considered when this drug is used in the diagnosis and treatment of adrenocorticoid-related diseases.