Liver Microsomes Of Wistar Rats Research Articles

Heating of vegetable oils influences the activity of enzymes participating in arachidonic acid formation in Wistar rats

Dietary intake of lipids and their fatty acids profile influence many aspects of health. Thermal processing changes the properties of edible oils and can also modify their metabolism, for example, eicosanoids formation. The aim of our study was to verify whether the activity of desaturases can be modified by lipids intake, especially by the fatty acids content. The experimental diets contained rapeseed oil, sunflower oil, and olive oil, both unheated and heated (for 10 minutes at 200 °C each time before administration), and influenced the fatty acids composition in serum and the activity of enzymes participating in arachidonic acid (AA) formation. The activity of desaturases was determined by measuring the amounts of AA formed in vitro derived from linoleic acid as determined in liver microsomes of Wistar rats. In addition, the indices of ∆(6)-desaturase (D6D) and ∆(5)-desaturase (D5D) have been determined. To realize this aim, the method of high-performance liquid chromatography has been used with ultraviolet-visible spectrophotometry detection. Diet supplementation with the oils rich in polyunsaturated fatty acids affects the fatty acids profile in blood serum and the activity of D6D and ∆(5)-desaturase in rat liver microsomes, the above activities being dependent on the kind of oil applied. Diet supplementation with heated oils has been found to increase the amount of AA produced in hepatic microsomes; and in the case of rapeseed oil and sunflower oil, it has also increased D6D activity.

Optimization, Validation and Application of Spectrophotometric Assay for 3-Hydroxy-3-methylglutarylcoenzyme A Reductase Activity

Purpose : To improve the sensitivity and specificity of spectrophotometric 3-hydroxy-3-methylglutarylcoenzyme A (HMG-CoA) reductase activity assay. Methods : Spectrophotometric HMG-CoA reductase detection in male Wistar rat liver microsomes was optimized by applying different conditions, such as reaction buffer pH, NADPH and protein concentration, and preincubation and reaction times. The optimal set of conditions was validated using HMG-CoA reductase inhibitors, namely, pravastatin, fluvastatin, and rosuvastatin. IC 50 was calculated and compared with that of a radiochemical assay. Ginkgo biloba extract’s (GBE50) inhibitory effect on HMG-CoA reductase activity was evaluated using the optimized spectrophotometric protocol. Results : The optimum assay conditions were as follows: reaction buffer pH 7.0, 100 μM NADPH, 50 μM HMG-CoA, and 200 μg/mL microsomal protein. The preincubation and reaction times were 20 and 60 min, respectively, at 37 oC. The IC50 of pravastatin, fluvastatin, and rosuvastatin under the optimum condition was 0.026, 0.015, and 0.007 μM while for radioisotope assay, it was 0.034, 0.049 and 0.0119 μM, respectively. GBE50 significantly inhibited HMG-CoA reductase activity in a concentrationdependent manner (p < 0.05). Conclusion : These results suggest that HMG-CoA reductase activity can be detected using the improved spectrophotometric assay. This assay can facilitate the discovery and development of new HMG-CoA reductase inhibitors in vitro. Keywords : Spectrophotometry, 3-Hydroxy-3-methylglutaryl-coenzyme A reductase activity, Cholesterol metabolism, Ginkgo biloba, Pravastatin, Fluvastatin, Rosuvastatin

HPLC-fluorescence Determination of EROD Activity in Wistar Rat Liver Microsomes Obtained by Two Different Extraction Procedures

HPLC-fluorescence Determination of EROD Activity in Wistar Rat Liver Microsomes Obtained by Two Different Extraction Procedures

HPLC-fluorescence Determination of EROD Activity in Wistar Rat Liver Microsomes Obtained by Two Different Extraction Procedures

This work determines the total protein concentration present in microsome fractions obtained by two distinct extraction procedures: ultracentrifugation and calcium aggregation. Additionally, the batch to batch variability within the extraction procedures used and the microsome stability and activity at -80°C during 90 days were determined using 7- ethoxyresorufin as a marker substract. An HPLC method to determine the activity of the cytochrome CYP450 1A1/2 was developed and fully validated by measuring 7-ethoxyresorufin-O-deethylase (EROD) activity by in vitro microsomal mixed-function biotransformation. The biotransformation product, resorufin, was obtained by incubation of 500 μg of rat liver microsomal proteins. The incubation time was 5 min and the assay was monitored by using an HPLC coupled to a fluorescence detector (λexc 530 nm and λem 582 nm). The total analysis time was 20 min and the sample preparation was a fast and simple protein precipitation. The results show that both extraction procedures are useful tools for the extraction of subcellular fractions allowing the recovery of appropriate protein concentration for further in vitro metabolism studies by cytochrome P450, specifically CYP1A1 and CYP1A2. Keywords: EROD activity, Calcium aggregation, Ultracentrifugation, Rat liver microsomes, HPLC-fluorescence, CYP1A1, CYP1A2, protein, ethoxyresorufin, microsomal

Glucuronidation of Mycophenolic Acid by Wistar and Mrp2-Deficient TR- Rat Liver Microsomes

In humans, mycophenolic acid (MPA) is metabolized primarily by glucuronidation in the liver to mycophenolate ether glucuronide (MPAGe) and mycophenolate acyl glucuronide (MPAGa). We have previously reported that in perfused livers of TR(-) rats (lacking the Mrp2 transporter), the clearance and hepatic extraction ratio of MPA were significantly lower compared with control Wistar rats, suggesting a difference in the capacity of the TR(-) rats to metabolize MPA in situ. There is very little information regarding the phase II metabolic capabilities of TR(-) rats; therefore, the aim of this study was to investigate the in vitro glucuronidation of MPA in Wistar and TR(-) rat liver microsomal protein. A second aim was to determine whether MPAGa, cyclosporine (CsA), and/or its metabolites AM1, AM1c, and AM9 inhibit the metabolism of MPA to MPAGe in rat liver microsomes. MPAGe formation rates by Wistar and TR(-) microsomes were 0.48 and 0.65 nmol/min/mg, respectively (p = 0.33). K(m) values for control and TR(-) microsomes were 0.47 and 0.50 mM, respectively (p = 0.81). The mean (S.E.M.) ratios of MPAGe formation by Wistar rat liver microsomes incubated with 50 microM MPA plus inhibitor versus 50 microM MPA alone were MPAGa 1.2 (0.1), CsA 0.7 (0.1) (p < 0.05), AM1 2.2 (0.3) (p < 0.05), AM1c 1.2 (0.2), and AM9 1.0 (0.2). Our results suggest that lower in situ glucuronidation of MPA in TR(-) rats may be because of inhibition of glucuronidation by endogenous and exogenous compounds that accumulate in the transporter-deficient rat. Whereas CsA inhibits glucuronidation of MPA, its metabolite AM1 enhances MPAGe formation by rat liver microsomes.

Role of rat liver cytochrome P450 3A and 2D in metabolism of imrecoxib1

To investigate the in vitro metabolism of imrecoxib in rat liver microsomes and to identify the cytochrome P450 (CYP) forms involved in its metabolism. Liver microsomes of Wistar rats were prepared using an ultracentrifuge. The in vitro metabolism of imrecoxib was studied by incubation with rat liver microsomes. To characterize the CYP forms involved in the 4 '-methyl hydroxylation of imrecoxib, the effects of typical CYP inducers (such as dexamethasone, isoniazid and beta-naphthoflavone) and of CYP inhibitors (such as ketoconazole, quinine, alpha-naphthoflavone, methylpyrazole, and cimetidine) on the formation rate of 4 '-hydroxymethyl imrecoxib were investigated. Imrecoxib was metabolized to 3 metabolites by rat liver microsomes: 4'-hydroxymethyl imrecoxib (M4), 4'-hydroxymethyl-5-hydoxyl imrecoxib (M3), and 4 '-hydroxymethyl-5-carbonyl imrecoxib (M5). Over the imrecoxib concentration range studied (5-600 micromol/L), the rate of 4'-methyl hydroxylation conformed to monophasic Michaelis-Menten kinetics. Dexamethasone significantly induced the formation of M4. Ketoconazole markedly lowered the metabolic rate of imrecoxib in a concentration-dependent manner. Moreover, a significant inhibitory effect of quinine on the formation of M4 was observed in microsomes obtained from control rats, isoniazid-induced rats, and b-naphthoflavone-induced rats. In contrast, a-naphthoflavone, cimetidine, and methylpyrazole had no inhibitory effects on this metabolic pathway. Imrecoxib is metabolized via 4'-methyl hydroxylation in rat liver microsomes. The reaction is mainly catalyzed by CYP 3A. CYP 2D also played a role in control rats, in isoniazid-induced rats and in beta-naphthoflavone-induced rats.

Strain Differences in CYP3A-Mediated C-8 Hydroxylation (1,3,7-Trimethyluric Acid Formation) of Caffeine in Wistar and Dark Agouti Rats: Rapid Metabolism of Caffeine in Debrisoquine Poor Metabolizer Model Rats

We observed significant strain differences [Dark Agouti (DA) > Wistar] in 1,3,7-trimethyluric acid formation (C-8 hydroxylation) during caffeine metabolism, though not in N-demethylations, in adult male DA and Wistar rats. In contrast, adult female and immature male rats of both DA and Wistar strains did not show significant differences in activity levels of C-8 hydroxylation. Kinetic studies using liver microsomes revealed that adult male DA rats have a larger Vmax for C-8 hydroxylation than do Wistar rats. Troleandomycin (TAO), known as a cytochrome P450 (CYP) 3A inhibitor, and an anti-rat CYP3A2 polyclonal antibody effectively reduced C-8 hydroxylation by rat liver microsomes in a concentration-dependent manner, suggesting that C-8 hydroxylation in rats is mediated largely by an isoform(s) of the CYP3A subfamily. Troleandomycin and the antibody did not inhibit the N-demethylations of caffeine by rat liver microsomes. Treatment of rats with CYP3A inducers caused a marked increase in C-8 hydroxylase activity. These results indicate that the rat CYP3A subfamily is capable of catalyzing C-8 hydroxylation of caffeine as is the case for human CYP3A4. The results of western blotting analysis using anti CYP3A antiserum showed that the staining intensity of the protein band in DA rat liver microsomes was higher than that in Wistar rat liver microsomes. We concluded that marked sex-dependent strain differences in C-8 hydroxylation of caffeine between Wistar and DA rats are due to the differences in the levels of expression of CYP3A in these strains of rats.

Strain differences in age-associated change in testosterone 6β-hydroxylation in Wistar and Dark Agouti rats

This study examines strain differences in testosterone (T)-hydroxylations between Wistar and Dark Agouti (DA) rats of both genders. The DA rat, an animal model, is a poor metabolizer of such drugs as debrisoquine, which are metabolized by cytochrome P450 (CYP) 2D. T-16α-, 2α-hydroxylations, which are linked to CYP2C11, were catalyzed at similar rates by the microsomes of both strains. In contrast, the liver microsomes from mature male DA rats catalyzed T-6β-hydroxylation, the CYP3A mediated activity, at higher rates (∼ 2-fold) than Wistar rat liver microsomes did. There was no difference between immature male DA and Wistar rats for T-6β-hydroxylation, indicating that the activity in male DA rat increases with maturation. Polyclonal antibodies raised against rat liver microsomal CYP3A2 and a CYP3A inhibitor, troleandomycin (TAO), effectively inhibited T-6β-hydroxylation by liver microsomes from both strains of rats. The level of T-6β- hydroxylation activity correlated well with the amount of CYP3A protein in the microsomes in mature as well as in immature male and female Wistar and DA rats. Northern blot analysis repeatedly indicated that the cellular contents of CYP3A2 mRNA are slightly (∼ 20%) higher in the liver of mature DA rats than in that of mature Wistar rats. These results indicate that the increased levels of CYP3A are responsible for the increased T-6β-hydroxylation activity and protein in DA rat.

Suppressive effect of interferon inducer, polyriboinosinic acid-polyribocytidylic acid on induction of uridine diphosphate-glucuronyltransferases and monooxygenases in liver microsomes of rats.

The effect of polyriboinosinic acid-polyribocytidylic acid [poly(I).poly(C)] on glucuronyltransferase activities toward 4-nitrophenol and 4-hydroxybiphenyl in liver microsomes of Wistar rats was examined by its single or co-administration with 3-methylcholanthrene and phenobarbital. The increased 4-nitrophenol glucuronyltransferase activity by treatment with 3-methylcholanthrene was significantly suppressed following the co-administration with poly(I).poly(C), although the activity was not affected by the treatment with poly(I).poly(C) alone. In addition, 4-hydroxybiphenyl glucuronyltransferase activity decreased or tended to decrease by the treatment with poly(I).poly(C) alone, and the activity induced by phenobarbital was strikingly decreased following the co-administration with poly(I).poly(C). This result suggested that poly(I).poly(C) comprehensively decrease the induction of glucuronyltransferases regardless of their multiple forms. Furthermore, contents of cytochromes P-450 and b5 were also decreased by the treatment with poly(I).poly(C) alone or the co-administration with the inducers. Concomitantly, arylhydrocarbon hydroxylase and benzphetamine N-demethylase activities were significantly decreased by the treatment alone or the co-administration with the inducers. These findings supported a view that the suppressive effect of poly(I).poly(C) may be derived from the prevention of de novo synthesis of the apoprotein of the enzymes and/or the increased degradation.

Congenital jaundice in rats due to the absence of hepatic bilirubin UDP-glucuronyltransferase enzyme protein

Three major UDP-glucuronyltransferase isoenzymes (50-54 kDa) have been identified by immunoblot analysis. Bilirubin UDP-glucuronyltransferase (54 kDa) was specially induced by treatment of the rats with clofibrate. This isoenzyme was not detectable in liver microsomal extracts from congenitally jaundiced Gunn rats and was not induced by treatment of these animals with clofibrate. Phenol UDP-glucuronyltransferase, the only isoenzyme determined to be present in foetal Wistar rat liver microsomes was not detected by enzyme assay or immunoblot analysis of foetal Gunn rat liver microsomal extracts. These results provide the first indication that bilirubin UDP-glucuronyltransferase and possible phenol UDP-glucuronyltransferase proteins are not present in the congenitally jaundiced Gunn rat.

Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes.

1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.

Delipidation and reactivation of UDP glucuronosyltransferase from rat liver

UDPglucuronosyltransferase was solubilized by treating Wistar rat liver microsomes with deoxycholate. Chromatography of this preparation on Bio-Gel P-30 resulted in extraction of 92% of phospholipids and complete loss of enzyme activity. UDPglucuronosyltransferase was reactivated by dialysing this delipidated preparation in the presence of lecithin, a mixture of liver microsomal lipids or microsomal preparations from livers of UDPglucuronosyltransferase-deficient Gunn rats. Virtually complete enzyme reactivation was obtained with regard to glucuronidation and glucosidation of bilirubin; however, the inactivation of UDPglucuronosyltransferase with p-nitrophenol as substrate was irreversible. These findings demonstrate that UDPglucuronosyltransferase with bilirubin as substrate is a lipid-requiring enzyme.