Anders Bergstrand

Isolation of rough and smooth microsomes from rat liver by means of a commercially available centrifuge

Abstract Cation-containing discontinuous sucrose gradients are commonly employed for the subfractionation of liver microsomes in various biochemical investigations. The procedure was originally described for the 60 rotor of the Christ Omega I centrifuge, but in most laboratories the isolation is performed by using the No. 30 or 40 rotors of Spinco centrifuges. It is point d out in this paper that a high cross-contamination occurs when fractionation is performed with these Spinco rotors. However, good separation with a high yield can be obtained with the Spinco centrifuge if the 40.2 rotor and the procedure that is described are employed.

Antidepressant-induced lipidosis with special reference to tricyclic compounds

Cationic amphiphilic drugs, in general, induce phospholipid disturbances. Tricyclic, as well as other antidepressants belong to this group. In experimental animals, antidepressants induce lipid storage disorders in cells of most organs, a so-called generalized phospholipidosis. This disorder is conveniently detected by electron microscopic examination revealing myelin figures. Myelin figures or myeloid bodies are subcellular organelles containing unicentric lamellar layers. The lipidotic induction potency during in vivo is related to the apolarity of the compound. Metabolism of phospholipids takes place within the cell continuously. Several underlying mechanisms may be responsible for the induction of the phospholipid disturbance. For instance, it has been suggested that the compounds bind to phospholipids and such binding may alter the phospholipids suitability as a substrate for phospholipases. Free TCA or metabolites thereof may also inhibit phospholipases directly, as has been demonstrated for sphingomyelinase in glioma and neuroblastoma cells. Both these mechanisms might result in phospholipidosis. Interaction between drug and phospholipid bilayer has been investigated by nuclear magnetic resonance technique. There seems to be large differences in the sensitivities amongst different organs. Steroid-producing cells of the adrenal cortex, testis and ovaries are in particular susceptible to drug-induced lipidosis. The so-called foam cells are lung macrophages located in the interstitium which become densely packed with myelin figures during TCA exposure. It requires about 3-6 weeks of treatment to develop this converted cell. In cell cultures however, phospholipidosis is demonstrated already after 24 h only. It appears that the cells that undergo TCA-induced lipidosis may recover after withdrawal of the drug. The time required to achieve complete recovery ranges from 3-4 weeks to several months, depending on the organ affected. Little is known about the functional significance of lipidosis. Even if TCA and other antidepressants show other effects, it has not been possible to exclusively relate it to phospholipidosis. However, few attempts have been made to correlate the physiological effects of TCAs in experimental animals to the morphological changes associated with phospholipidosis. There is an increasing evidence however, that cationic amphiphilic drugs may have effects on immune function, signal transduction and receptor-mediated events, effects that to some extent might be related to disturbances in phospholipid metabolism.

Preparation and characterization of subcellular fractions from the liver of C57B1/6 mice, with special emphasis on their suitability for use in studies of epoxide hydrolase activities

The present study was designed to prepare and characterize subcellular fractions from the liver of male C57B1/6 mice, with special emphasis on their suitability for use in studies of epoxide hydrolase isozymes. The effects of different washing and pelleting procedures on the mitochondrial, microsomal and cytosolic fractions were studied. It was found that 133,000 gav for 60 min (i.e. more extensive force than the usual 105,000 gav for 60 min) was necessary to obtain a membrane-free cytosolic fraction, while one wash for microsomes and two washes for mitochondria yielded reasonably pure fractions. The purity of the different fractions obtained by differential centrifugation was then determined using established enzyme markers and morphological examination with the electron microscope. Several enzymes involved in drug metabolism were also measured in these fractions. The subcellular distributions obtained here for marker enzymes closely resemble those reported for rat liver. Starvation had no significant effect on the epoxide hydrolase activities nor did the addition of mouse bile or rat liver cytosol, which might contain inhibitors. The change in epoxide hydrolase activities with time after preparation of the subcellular fractions was studied, as well as the effect of freeze-thawing. The subfractions prepared here are suitable for the further characterization of the different forms of epoxide hydrolase present in mouse liver, as well as for other studies requiring well-characterized subfractions.

Effects of perfluorooctanoic acid : a potent peroxisome proliferator in rat : on Morris hepatoma 7800C1 cells, a rat cell line

In this study, Morris hepatoma 7800C1 cells (from rat) were exposed to 500 microM perfluorooctanoic acid (PFOA) in the culture medium for 7 days. This treatment resulted in inductions of catalase, lauroyl-CoA oxidase (which catalyzes the first step in peroxisomal beta-oxidation) and of cytochrome P-450IVA (specialized for omega- and omega-1 hydroxylation of fatty acids). Northern blot analysis revealed that the level of mRNA for peroxisomal fatty acyl-CoA oxidase was enhanced in cells treated with PFOA. Inductions of the enzymes mentioned above are generally connected with peroxisome proliferation in vivo. This work also includes a comparison between the activities of catalase, lauroyl-CoA oxidase, DT-diaphorase and glutathione transferase in rat liver homogenate and 7800C1 cells in order to investigate to what extent this cell line differs from the situation in vivo. The findings suggest that the cells selectively lost most of their peroxisomes during transformation into a cell line and subsequent propagation. The control activities of catalase and lauroyl-CoA oxidase (marker enzymes for peroxisomes) were only about 2% of the corresponding enzyme activities in rat liver. In addition, a morphological study revealed that the frequency of peroxisomes in 7800C1 cells is very low. The control activity of glutathione transferase in 7800C1 cells was 11% of the corresponding activity in rat liver homogenate, whereas the level of DT-diaphorase was virtually the same in 7800C1 cells as in rat liver. Electron microscopic investigation of the control cultures revealed all signs of viable cells, with well-developed cell organelles. Treatment of 7800C1 cells with 500 microM PFOA has little effect on cellular morphology.

Hepatic peroxisome proliferation in vitamin A-deficient mice without a simultaneous increase in peroxisomal acyl-CoA oxidase activity.

Vitamin A-adequate and vitamin A-deficient C57B1/6 mice were treated for ten days with 0.02% (w/w) perfluorooctanoic acid (PFOA) in their diet. Treated vitamin A- adequate and -deficient mice demonstrated approximately the same increases in liver somatic index (g liver/g body weight) (somewhat more than 2-fold) and mitochondrial protein content (5-fold). PFOA treatment resulted in a 26-fold increase in hepatic peroxisomal lauroyl-CoA oxidase activity in vitamin A-adequate mice, whereas the same activity was unchanged in vitamin A-deficient mice. Vitamin deficiency itself caused a 3- to 4- fold increase in cytosolic catalase activity and a smaller increase in the activity of microsomal cytochrome P-450 IVA (lauric acid omega- and omega-1 hydroxylase) in this same organ. The induction of the activities of these enzymes was less prominent in vitamin A-deficient mice compared with the effect caused by PFOA in vitamin A-adequate mice, resulting in approximately the same maximal values for these parameters in both groups (i.e approx 21 micromol/g liver - min and 350 nanomol/g liver - min, respectively). A 70 kDa protein, presumable the multifunction protein, was shown by Commassie blue staining of SDS-polyacrylamide gels and by immunoblotting (with antibodies towards the multifunctional protein) to be induced to approximately the same degree in vitamin A-adequate and deficient mice. A morphometric study revealed that PFOA causes the same extent of hepatic peroxisome proliferation in vitamin A-deficient as in vitamin A-adequate mice. The possibility that PFOA exerts its effect in vivo through at least two different mechanisms is discussed.

Preparation and characterization of subcellular fractions from the liver of the northern pike, Esox lucius

The present study was designed to prepare and characterize subcellular fractions from the liver of the Northern pike (Esox lucius), with special emphasis on the preparation of microsomal fractions suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation, as well as the recovery of different organelles, was determined using both enzyme markers and morphological examination with the electron microscope. Attempts were also made to increase the recovery of fragments of the endoplasmic reticulum in the microsomal fraction. Finally, the subcellular distribution of several drug-metabolizing enzymes (cytochrome P-450, benzpyrene monoxygenase, epoxide hydrolase and glutathione transferases) were determined. With the exception of the subcellular distribution of epoxide hydrolase, the results obtained here resemble closely those reported fo rat liver and the microsomal fraction prepared is highly suitable for further studies of drug metabolism in pike liver.

Preparation and characterization of subcellular fractions suitable for studies of drug metabolism from the trunk kidney of the northern pike (Esox lucius) and assay of certain enzymes of xenobiotic metabolism in these subfractions

The present study was designed to prepare and characterize subcellular fractions from the trunk kidney of the Northern pike (Esox lucius), with special emphasis on the preparation of a microsomal fraction suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation, as well as the recovery of different organelles, was determined using both enzyme markers and morphological examination with the electron microscope. Finally, the subcellular distributions of several drug-metabolizing enzymes (NADPH-cytochrome c reductase, NADH-ferricyanide reductase, glutathione transferase, epoxide hydrolase) were determined. With the exception of NADPH-cytochrome c reductase, the subcellular distributions obtained here for drug metabolizing and marker enzymes closely resembled those reported for rat liver. NADPH-cytochrome c reductase was apparently partially solubilized here from microsomal vesicles by an endogenous protease, which reduced its usefulness as a marker enzyme and raises questions concerning the measurement of activities catalyzed by the cytochrome P-450 system in these subfractions. In other respects the microsomes and supernatant fraction prepared here from the trunk kidney of the pike seem to be as well suited for investigations of drug metabolism as are the corresponding fractions from rat and pike liver.

Preparation and characterization of subcellular fractions from the intestinal mucosa of the Northern pike (Esox lucius), with special emphasis on enzymes involved in xenobiotic metabolism

Abstract The present study was designed to prepare and characterize subcellular fractions from the intestinal mucosa of the Northern pike (Esox lucius), with special emphasis on the preparation of a microsomal fraction suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation, as well as the recovery of different organelles, was determined using both enzyme markers and morphological examination with the electron microscope. The subcellular distributions of several enzymes involved in drug metabolism (NADPH-cytochrome c reductase, NADH-ferricyanide reductase, epoxide hydrolase activity towards both cis- and trans-stilbene oxide as substrates, and glutathione transferase) were also examined. The subcellular distributions obtained here for drug-metabolizing and marker enzymes closely resembled those reported for rat and pike liver. The microsomal fraction obtained contained about 50% of the total endoplasmic reticulum. This fraction was relatively free of nuclei, mitochondria, Golgi, peroxisomes and cytosol, but relatively heavily contaminated with lysosomes and fragments of the plasma membrane. Within the limitations discussed, the subfractions prepared here are suitable for further characterization of drug-metabolizing systems in the intestinal mucosa of the Northern pike, as well as for other studies with this tissue.

Preparation and characterization of subcellular fractions from the head kidney of the Northern pike (Esox lucius), with particular emphasis on xenobiotic-metabolizing enzymes.

The present study was designed to prepare and characterize subcellular fractions from the head kidney of the Northern pike (Esox lucius), with special emphasis on the preparation of a microsomal fraction suitable for studying xenobiotic metabolism. The purity of the different fractions obtained by differential centrifugation as well as the recovery of different cell components was determined using both enzyme markers and morphological criteria. Finally, the subcellular distributions of several drug-metabolizing enzymes (NADPH-cytochrome c reductase, NADH-ferricyanide reductase, glutathione transferase, epoxide hydrolase) were determined. With the exception of NADPH-cytochrome c reductase, the subcellular distributions obtained here for drug-metabolizing and marker enzymes closely resembled those reported for rat liver. NADPH-cytochrome c reductase was apparently partially solubilized here from microsomal vesicles by an endogenous protease, which reduced its usefulness as a marker enzyme and raises questions concerning the measurement of activities catalyzed by the cytochrome P-450 system in these subfractions. In other respects the microsomal fraction prepared here from the pike head kidney seems well-suited for studies of drug metabolism.

The involvement of selenium in peroxisome proliferation caused by dietary administration of clofibrate to rats

The effects of dietary treatment with clofibrate (0.5% w/w for 10 days) on the livers of selenium-deficient male rats were examined. The peroxisome proliferation (as determined by electron microscopy) in the livers of selenium-deficient animals was much less pronounced than in the case of selenium-adequate rats and no increase in peroxisomal fatty acid beta-oxidation (assayed both as antimycin-insensitive palmitoyl-CoA oxidation and lauroyl-CoA oxidase activity) was observed in the deficient animals. On the other hand, in selenium-deficient rats clofibrate caused increases in the specific activity of microsomal lauric acid omega- and omega-1-hydroxylation and an apparent change in mitochondrial size, seen as a redistribution of mitochondria from the 600 x g(av) pellet to the 10,000 x g(av) pellet, which were approximately 50% as great as the corresponding effects on control animals. Obviously, then, these three different effects of clofibrate are not strictly coupled and may involve at least partially distinct underlying mechanisms. Initial experiments demonstrated that peroxisome proliferation could be obtained by exposing primary hepatocyte cultures derived from selenium-deficient rats to clofibric acid (an in vivo hydrolysis product of clofibrate which is the proximate peroxisome proliferator), nafenopin or mono(2-ethylhexyl)phthalate. This finding suggests that selenium deficiency does not have a direct influence on the basic process(es) underlying peroxisome proliferation, but rather has indirect effects, influencing, for example, the pharmacokinetics of clofibrate and/or hormonal factors.