Microsomal Suspensions Research Articles

Lipid peroxidation in rat liver microsomes during naproxen metabolism

Naproxen, a non-steroidal anti-inflammatory drug, is known to be highly effective and relatively safe, but some side-effects in the liver have been reported. In the present study, the effect of naproxen metabolism on rat liver microsomes was studied by determining lipid peroxidation in terms of thiobarbituric acid reactive substances (TBA-RS), high molecular weight protein aggregates and fluorescent substances formed in the microsomal suspension containing naproxen, NADPH and MgCl 2. Lipid peroxidation was found to occur at 10 mM naproxen. Production of chemiluminescence from the microsomal suspension was observed during naproxen metabolism. The time course of 6-demethylnaproxen formation by O-demethylation of naproxen appeared to be comparable to that of the chemiluminescence production in their initial periods of production. These results suggest that the lipid peroxidation was provoked through the reactive oxygen species generated during the oxidative metabolism of naproxen.

Rat brain microsome fluidity as modified by prenatal ethanol administration.

The fluorescence anisotropy (r) of diphenylhexatriene (DPH) and of trimethylamino-diphenylhexatriene (TMA-DPH) as a function of temperature (10 degrees to 54 degrees C) was measured in brain microsomes of newborn rats prenatally exposed to ethanol. In this temperature range, the relationship between r and T was linear. The addition of ethanol in vitro to microsomal suspensions influenced the slope of the line of r versus T only when DPH was used as a probe and with high concentrations of the alcohol (> or = 0.3 M). The administration of ethanol (18% of total energy intake) in vivo to pregnant dams affected the slope of the lines of r versus T of the microsomes of pups, either using DPH or TMA-DPH as probes. The slope was also affected in brain microsomes obtained from dams, yet, only with TMA-DPH and in the opposite sense than in pups. We conclude that the effect of prenatal exposure to ethanol depended on metabolic alterations induced by the alcohol and not on its detergent properties for the following reasons: (a) The effects in vitro and in vivo were different and (b) in vitro effects could be obtained only with high concentrations (> or = 0.3 M), whereas in vivo effects were produced by small doses of ethanol. Besides, the effects of the administration of the alcohol in vivo were different in adult and intrauterine life.

An extract of Desmodium adscendens inhibits NADPH‐dependent oxygenation of arachidonic acid by kidney cortical microsomes

AbstractCortical microsomes of human kidneys were used to study the effect of an extract of Desmodium adscendens on the cytochrome P450‐dependent arachidonic acid (AA) metabolism. The phenolic compounds tyramine and hordenine, as well as triterpenoid saponins known to be present in the plant were also evaluated along with the extract, an n‐butanol fraction (nBF) of the crude plant extract. The extract and the compounds were pre‐incubated with the microsomal suspension before the addition of cofactors and radioactive AA. The products of the reactions and the unreacted AA were extracted, separtaed by HPLC and measured radiometrically. The total amount of radioactive AA converted, and the production of AA metabolites identified as epoxy‐eicosatrienoic acids, monohydroxy‐eicosatetraenoic acids, 19‐, 20‐ and di‐hydroxy derivatives of AA, were inhibited by nBF. The inhibition by nBF was shown to be dose‐dependent. A saponin, sapogenin and the phenolic compounds present in D. adscendens did not inhibit this oxygenation of AA. The results indicate that this plant contains an as yet unidentified inhibitor for the third pathway of AA metabolism.

Alteration of Δ-6 desaturase by vitamin E in rat brain and liver

delta-6 Desaturase, measured at substrate saturation using linoleic acid, was found to be increased by more than two-fold when the content of vitamin E in brain microsomal membrane suspension was increased (up to 7.5 micrograms/mg membrane protein, i.e. 100 micrograms/g tissue from which microsomes were prepared). In contrast, this activity was reduced by 25% in the liver. This raises the question of the multiple role of vitamin E in membranes, the control of membrane polyunsaturated fatty acids through synthesis, and their protection against peroxidation.

Effect of cadmium on ciliary and ATPase activity in the gills of freshwater mussel Anodonta cygnea

1. Cadmium (≤ 50 μM) decreases the heat resistance (39°C) of the activity of frontal cilia in the Anodonta cygnea gills incubated in dechlorinated tap water, while in the presence of added 2 mM Ca2+ the minimal acting concentration of cadmium rises up to 100 μM. 2. The inhibitory effect of Cd2+ (1.5 mM) on the ATPase activity measured in the gill microsomal fraction is temperature dependent and increases as follows: ouabain insensitive Na2+- or K+-ATPase (no inhibition), Ca2+-ATPase (50% inhibition), Mg2+-ATPase (100% inhibition). 3. Cadmium itself (≤ 50 μM) added to microsomal suspension stimulates the H+-sensitive ATP hydrolysis resembling on its pH-dependence the Mg2+- but not Ca2+-ATPase activity. 4. Cd2+ can mimic the effect of Mg2+ as a cofactor required for activation of the ouabain-insensitive Na+- or K+-ATPase. Monovalent cations fail to activate the ATPase when Mg2+ is substituted by Ca2+. 5. One of the mechanisms underlying the toxicity of Cd2+ to Anodonta gills could be based upon an interaction of Cd2+ with Mg2+-ATPase followed by suppression of the ciliary activity.

Evaluation of adriamycin-induced lipid peroxidation

Lipid peroxidation is known to be a mechanism for Adriamycin®-induced toxicity. In the present study, two methods which detect fluorescent substances and high molecular weight protein aggregates in peroxidized membranes were applied to Adriamycin-induced lipid peroxidation in liver microsomes. A rat liver microsomal suspension containing an NADPH-generating system was incubated with Adriamycin. Thiobarbituric acid reactive substances (TBA-RS), formed during this incubation, were transferred from the microsomes to the medium. Fluorescent substances determined by the fluorescence emitted from both the microsomes themselves and the chloroform/methanol extracts of the microsomes, were found to be formed during this incubation. High molecular weight protein aggregates determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, were also formed. Fluorescent substances and high molecular weight protein aggregates were found in microsomal membranes themselves and increased time dependently. These substances retained in membranes can be of great use to delineate the site of Adriamycin-induced lipid peroxidation in vitro and in vivo and to determine how this lipid peroxidation affects the membrane.

Photodynamic effects of chloroaluminum phthalocyanine tetrasulfonate are mediated by singlet oxygen: In vivo and in vitro studies utilizing hepatic microsomes as a model membrane source

Chloroaluminum phthalocyanine tetrasulfonate (Al-PcTS) is a promising photosensitizer for the photodynamic therapy (PDT) of cancer. In this study, we investigated the in vivo and in vitro photodestruction of hepatic microsomal membranes by AlPcTS and studied the role of reactive oxygen species in this process. Irradiation of hepatic microsomes prepared from AlPcTS-pretreated SENCAR mice to ≈675 nm light resulted in rapid destruction of cytochrome P450 and associated monooxygenase activities, and enhancement of lipid peroxidation in a light-dose-dependent manner. The specificity of AlPcTS and light dependency on photodestruction of microsomal membranes was confirmed by Western blot analysis. Similar results were obtained when AlPcTS was added in vitro to a suspension of hepatic microsomes prepared from control animals followed by irradiation to ≈675 nm light. Among the quenchers of singlet oxygen, superoxide anion, hydrogen peroxide, and hydroxyl radical, only the quenchers of singlet oxygen such as sodium azide, histidine, and 2,5-dimethyl furan afforded substantial protection in a dose-dependent manner against AlPcTS-mediated photodestruction of cytochrome P450 and associated monooxygenase activities, and photoenhancement of lipid peroxidation under both in vivo and in vitro conditions. These results suggest that lipid-rich microsomal membranes may be the potential targets of cell injury by AlPcTS-based PDT and that this process is mediated by singlet oxygen.

Studies on toxicological mechanisms of gas-phase cigarette smoke and protection effects of GTP

Gas-phase cigarette smoke (GPCS) was able to induce lipid peroxidation in lecithin liposomes, rat liver microsomes, and rat lung cells (RLC), and change the membrane fluidity of RLCs. Lipid free radicals were trapped in a GPCS-treated microsomal suspension by using 4-POBN as the spin trap. In addition, it was found that GPCS-peroxidized liposomes in appropriate degree of lipid peroxidation had the ability to increase the generation of superoxide anions in rat peritoneal neutrophils (RPN). Effects of green tea polyphenols (GTP) on the GPCS-induced damages were investigated The results showed that GTP was capable of inhibiting the GPCS-induced damages.

Studies on chemical protectors against radiation. XXXV. Effects of radioprotective Chinese traditional medicines on radiation-induced lipid peroxidation in vivo and in vitro.

The fluctuation of lipid peroxidation (LP) in 9 tissues was investigated in mice for 7 d after whole-body X-irradiation with a lethal dose of bone marrow death. LP increased significantly in bone marrow, thymus, spleen and liver following irradiation, and slightly in brain and testis, but not in blood plasma, submaxillary gland or kidney. The effects of 7 radioprotective Chinese traditional medicines (CTMs) and cysteamine (MEA) on the radiation-induced LP in 4 tissues were studied by i.p. injection before or after irradiation and their LP content in tissues was measured 2 d after irradiation. Most CTMs showed significant inhibition of radiation-induced LP in bone marrow and liver, especially when injected prior to irradiation. Some CTMs also showed such inhibition in spleen. MEA only inhibited the increase of LP in liver when injected before irradiation, but enhanced the increase of LP in spleen. None of these radioprotectors including MEA was recognized to inhibit radiation-induced LP in thymus. The in vitro experiments were carried out using mouse liver microsomal suspensions (MS). The MS were prepared from normal (non-irradiated) mice. Each of the 8 radioprotectors was added to MS before or after irradiation and then post-irradiation-incubated at 37 degrees C. All markedly inhibited radiation-induced LP if added before irradiation, but were slightly less effective if added after.

Hepatic EROD activity in dab Limanda limanda in the German Bight using an improved plate-reader method

During the Bremerhaven Workshop an improved, fast and easy method for the determination of ethoxyresorufin-O-deethylase (ERODl activity in dab Limanda limanda liver was tested. The improvement involved the use of a 12500 x g supernatant instead of the microsomal fraction, thus onutling the need for long ultra-centrifugations which cannot be carried out aboard a research vesse!. EROD activities in the 12500 x g supernatant and in the microsomal suspension were weil correlated. Despite its disadvantages of lower specific EROD activity, higher minimum detection limit and higher analytical variance, the 12500 x g supernatant \Vas used for further analysis. EROD activity in dab liver showed a spatial trend corresponding to a pollution gradient in the German Bight EROD activity was highest at sampling sites near to the Elbe and Weser estuaries. The same trend was found in liver concentrations of PCB and lindane, but not of heavy metals and other pesticides.

Detection of the Ethyl-and Pentyl-Radical Adducts of α-(4-Pyridyl-1-Oxide)-N-Ter J-Butylnitrone in Rat-Liver Microsomes Treated with ADP, NADPH and Ferric Chloride

HPLC-EPR analyses of the reaction mixtures of microsomal suspensions incubated with ADP, ferric chloride, NADPH and alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were performed. In the elution pattern of the reaction mixture, three peaks (peaks 1, 2 and 3) were detected. The radical adducts (1 and 3) were identified as being the pentyl- and ethyl-radical adducts of 4-POBN by comparing their retention times with those of the authentic radical adducts.

A chelator is required for microsomal lipid peroxidation following reductive ferritin-iron mobilisation.

In the past, antioxidant and chelator studies have implicated a role for iron-dependent oxidative damage in tissues subjected to ischaemia followed by reperfusion. As ferritin is a major source of iron in non-muscular organs and therefore a potential source of the iron required for oxygen radical chemistry, we have determined conditions under which ferritin iron reduction leads to the formation of a pool of iron which is capable of catalysing lipid peroxidation. Under anaerobic conditions and in the presence of rat liver microsomes, flavin mononucleotide (FMN) catalysed the reduction of ferritin iron as shown by both continuous spectrophotometric measurements of tris ferrozine-Fe(II) complex formation and post-reaction Fe(II) determination. The presence of either ferrozine or citrate was not found to alter the time course or extent of ferritin reduction. In contrast, the addition of air to the reactants after a 20 min period of anaerobic reduction resulted in peroxidation of the microsome suspension (as determined with the 2-thiobarbituric acid test) only in the presence of a chelator such as citrate, ADP or nitrilotriacetic acid. These results support the concept that reduced ferritin iron can mediate oxidative damage during reperfusion of previously ischaemic tissues, provided that chelating agents such as citrate or ADP are present.

Thyroid Hormones and Glucocorticoids Act Synergistically in the Regulation of the Low Affinity Glucocorticoid Binding Sites in the Male Rat Liver*

The low affinity glucocorticoid binding sites (LAGS) have been described and partially characterized in both the nuclei and microsomes of rat liver. The LAGS concentration is under endocrine regulation, as proved by their decrease after adrenalectomy and their almost complete disappearance after hypophysectomy. This article describes new data that also implicate the thyroid hormones in the endocrine regulation of LAGS. The LAGS were measured by [3H]dexamethasone exchange assay in crude microsome suspensions of rat liver. Propylthiouracil-induced hypothyroidism (TX) provoked a 90% reduction in the LAGS levels with respect to the control value. The administration of T3 to TX rats was able to completely restore the LAGS level. On the other hand, adrenalectomy (ADX) provoked a 50% decrease in LAGS levels, and this effect could be reverted by treatment with corticosterone acetate. TX rats that were also adrenalectomized (TX-ADX) showed a LAGS level similar to that of the TX rats. However, treatment of these rats with T3 was much less effective than in TX rats. A complete restoration of the LAGS level in TX-ADX rats could be achieved only with a combined treatment of corticosterone acetate plus T3. Similar results to those obtained in TX-ADX rats were also obtained in immature or hypophysectomized rats, two experimental models known to possess very low or undetectable levels of LAGS. From these findings we conclude that: 1) thyroid hormones, as well as glucocorticoids, play an important role in the regulation of the LAGS level; 2) glucocorticoids and thyroid hormones act synergistically in the endocrine regulation of LAGS; and 3) the results obtained in the hypophysectomized rats point to a direct action of glucocorticoids and T3 on the LAGS level of the rat liver.

Effects of added protein (bovine serum albumin) on the rate and enantiotopic selectivity of oxidative O-demethylation of methoxychlor in rat liver microsomes

The addition of bovine serum albumin (BSA) to rat liver microsomal suspensions during the O-demethylation of methoxychlor (1,1-bis(4-methoxyphenyl)-2,2,2-trichloroethane) produced a good substrate concentration-reaction rate relationship, and allowed us to determine the rate parameters for this reaction. The apparent K m became larger when the albumin content was increased, whereas V max was not changed. The enantiotopic selectivity of the reaction dramatically changed when the substrate concentration was increased in the absence of BSA in phenobarbital-treated rat liver microsomes.

Metabolic activation of 1,2,4-trichlorobenzene and pentachlorobenzene by rat liver microsomes: A major role for quinone metabolites

Microsomal metabolism of 1,2,4-[ 14 C] trichlorobenzene (1,2,4-TrCB) and [ 14 C] pentachlorobenzene (PeCB) was studied with special emphasis on the conversion-dependent covalent binding to protein and DNA. 1,2,4-TrCB was metabolized to 2,3,6- and 2,4,5-trichlorophenol, and to a lesser extent to 2,4,6- and 2,3,5-trichlorophenol, and trichlorohydroquinone. About 10% of all metabolites became covalently bound to protein in a rather nonselective way. For 1,2,4-TrCB and PeCB a strong correlation between secondary metabolism to hydroquinones and covalent binding was established. Protein binding was completely inhibited by the addition of ascorbic acid, indicating quinone metabolites as the sole reactive species formed. Both 1,2,4-TrCB and PeCB alkylated DNA, although to a much lesser extent than protein (0.5 and 0.3% of all metabolites, respectively). Nonquinone intermediates, presumably epoxides, were responsible for a minor portion of the observed DNA binding, since complete inhibition by ascorbic acid was not reached. The differential role of cytochrome P450 both in primary and in secondary metabolism was demonstrated by the use of microsomes from rats pretreated with different inducers. Dexamethasone (DEX) microsomes (cytochrome P450IIIA1) showed the highest activity toward these chlorinated benzenes (14 nmol/mg/5 min for 1,2,4-TrCB and 36 nmol/mg/10 min for PeCB), both with regard to the formation of phenols and to the formation of protein-bound metabolites. In addition, DEX microsomes preferentially formed 2,3,6-trichlorophenol, whereas other microsomal suspensions formed 2,4,5-trichlorophenol as the major isomer. The present study clearly demonstrates the high alkylating potency of secondary quinone metabolites derived from chlorinated benzenes and poses a need for reevaluation of the role of epoxides in the observed toxicity of these compounds.

Effect of calcium channel blocking agents on the reductive metabolism of halothane

The effect of calcium channel blocking agents on the reductive metabolism of halothane in liver microsomes of guinea pigs was investigated. The reaction mixture for the measurement of the end products consisted of microsomal suspension, 5 mM NADPH, calcium channel blocking agents (verapamil, diltiazem, nicardipine and nifedipine) and halothane in 0.1 M phosphate buffer (pH 7.4). The reductive metabolism of halothane was inhibited competitively by verapamil, diltiazem and nicardipine. The binding spectra for the interaction of these three drugs with cytochrome P-450 in microsomes were investigated. Verapamil caused the reverse type I difference spectrum and diltiazem caused the type I difference spectrum. However, the change caused by nicardipine was not observed by the presence of its specific spectra. NADPH-cytochrome P-450 reductase activity in microsomes did not change by the addition of these three drugs. These results suggest that these three calcium channel blocking agents inhibit the production of radical intermediates during the reductive metabolism of halothane.

Medicinal azides. Part 8. The in vitro metabolism of p-substituted phenyl azides

1. A series of p-substituted aromatic azides was synthesized and their metabolism investigated in suspensions of mouse liver microsomes and mouse hepatocytes. Metabolite analysis was performed by h.p.l.c. 2. On incubation with microsomes under anaerobic conditions p-nitro, p-cyano- and p-chlorophenyl azide afforded metabolites which co-chromatographed with the respective aromatic amines. The rate at which p-nitrophenyl azide was metabolically reduced was approximately 20-fold that observed for p-cyano- and p-chlorophenyl azide. 3. Phenyl azide, p-methoxyphenyl azide and the aliphatic congener, phenethyl azide, did not furnish detectable amounts of metabolites on incubation with microsomes under anaerobic conditions. When phenyl azide and p-methoxyphenyl azide were incubated with hepatocytes or microsomes under aerobic conditions the resulting chromatograms furnished peaks which co-eluted with authentic p-hydroxyphenyl azide. 4. The microsomal reduction of p-nitrophenyl azide was dependent upon the presence of viable microsomes and NADPH, and on the absence of oxygen above the incubation medium.

Role of microsomal cytochrome P-450 in the formation of ecdysterone in larval house fly

1. 1. Six cytochrome P-450 species have been purified to varying extents from microsomes obtained from ecdysone-induced house fly larvae by the use of octylamino Sepharose-4B, Synchropak AX-300, Synchropak CM-300 and TSK-DEAE-5 PW column chromatography. 2. 2. One of the fractions apparently corresponded to a mixture of low- and high-spin cytochrome P-450 as judged by spectral characteristics. 3. 3. Molecular weights of the cytochrome P-450 species ranged from 50,000 to 57,000. 4. 4. In a reconstituted system, all the microsomal species hydroxylated ecdysone at rates within the range of microsomal suspensions, as it occurs with mitochondrial fractions 1, 2, 3, 5, and 6 (Srivatsan et al., 1990, Biochem. biophys. Res. Commun. 166, 1372–1377); whereas, mitochondrial fraction 4 hydroxylates ecdysone at significantly higher rates. 5. 5. It is postulated that the 20-monooxygenation of ecdysone is a mitochondrial event which requires the induction of a low- K m cytochrome P-450 species by ecdysone. 6. 6. Microsomal hydroxylation of ecdysone may not be of physiological significance, as K m values for the reaction are above the normal concentrations of the hormone and the activity is not inducible by ecdysone (Agosin et al., 1988, Arch. Insect Biochem. Physiol. 9, 107–117).

Expression of human liver cytochrome P450 IIIA4 in yeast. A functional model for the hepatic enzyme

Cytochrome P-450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium-channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage lambda gt11 expression library [Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S. & Guengerich, F. P. (1986) Proc. Natl Acad. Sci. USA 83, 8064-8068]. We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8-2 [Cullin, C. & Pompon, D. (1988) Gene 65, 203-217]. Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous-CO spectrum typical of cytochrome P-450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450-5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8-2 alone), as revealed by immunoblotting with anti-P450-5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8-10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast-expressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast-expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4-oxidation, quinidine 3-hydroxylation and N-oxygenation, and N-demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH-cytochrome P-450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPH-cytochrome P-450 reductase increased the oxidation rates. Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P-450 (lanosterol 14-demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P-450 (30 times less). Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P-450.

Evidence for the involvement of singlet oxygen in the photodestruction by chloroaluminum phthalocyanine tetrasulfonate

In recent years, choloroaluminum phthalocyanine tetrasulfonate (A1PCTS) has been shown to be a promising photosensitizer for the photodynamic therapy (PDT) of cancer. Although its mechanism of photodynamic action is not well defined, A1PCTS is going to be under clinical trials of PDT. In this study, in vitro addition of A1PCTS to a suspension of rat epidermal microsomes followed by irradiation with red light (approximately 675 nm) resulted in significant destruction of cytochrome P-450 and associated monooxygenase activities. The photodestructive effect was dependent on both the dose of A1PCTS and the duration of light exposure. Studies using various quenchers of reactive oxygen species showed that only scavengers of singlet oxygen such as histidine, 2,5-dimethylfuran, beta-carotene and sodium azide afforded substantial protection against photodestruction. Our data indicate the direct involvement of singlet oxygen in the A1PCTS-mediated photodestructive process.