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Anesthesiology Aug 2001Halothane is extensively (approximately 50%) metabolized in humans and undergoes both oxidative and reductive cytochrome P450-catalyzed hepatic biotransformation....
BACKGROUND
Halothane is extensively (approximately 50%) metabolized in humans and undergoes both oxidative and reductive cytochrome P450-catalyzed hepatic biotransformation. Halothane is reduced under low oxygen tensions by CYP2A6 and CYP3A4 in human liver microsome to an unstable free radical, and then to the volatile metabolites chlorodifluoroethene (CDE) and chlorotrifluoroethane (CTE). The free radical is also thought to initiate lipid peroxidation. Halothane-dependent lipid peroxidation has been shown in animals in vitro and in vivo but has not been evaluated in humans. This investigation tested the hypothesis that halothane causes lipid peroxidation in human liver microsomes, identified P450 isoforms responsible for halothane-dependent lipid peroxidation, and tested the hypothesis that lipid peroxidation is prevented by inhibiting halothane reduction.
METHODS
Halothane metabolism was determined using human liver microsomes or cDNA-expressed P450. Lipid peroxidation was quantified by malondialdehyde (MDA) formation using high-pressure liquid chromatography-ultraviolet analysis of the thiobarbituric acid-MDA adduct. CTE and CDE were determined by gas chromatography-mass spectrometry.
RESULTS
Halothane caused MDA formation in human liver microsomes at rates much lower than in rat liver microsomes. Human liver microsomal MDA production exhibited biphasic enzyme kinetics, similar to CDE and CTE production. MDA production was inhibited by the CYP2A6 inhibitor methoxsalen but not by the CYP3A4 inhibitor troleandomycin. Halothane-dependent MDA production was catalyzed by cDNA-expressed CYP2A6 but not CYP3A4 or P450 reductase alone. CYP2A6-catalyzed MDA production was inhibited by methoxsalen or anti-CYP2A6 antibody.
CONCLUSIONS
Halothane causes lipid peroxidation in human liver microsomes, which is catalyzed by CYP2A6, and inhibition of halothane reduction prevents halothane-dependent lipid peroxidation in vitro.
Topics: Anesthetics, Inhalation; Animals; Aryl Hydrocarbon Hydroxylases; Cytochrome P-450 CYP2A6; Cytochrome P-450 Enzyme System; Halothane; Humans; In Vitro Techniques; Isoenzymes; Lipid Peroxidation; Malondialdehyde; Microsomes, Liver; Mixed Function Oxygenases; Oxidation-Reduction; Rats
PubMed: 11506127
DOI: 10.1097/00000542-200108000-00037 -
Journal of B.U.ON. : Official Journal... 2015The purpose of this study was to determine possible genotoxic effects of a new very promising antibacterial/antiviral drug FS-1.
PURPOSE
The purpose of this study was to determine possible genotoxic effects of a new very promising antibacterial/antiviral drug FS-1.
METHODS
The drug was tested in TA98, TA100, TA102, TA1535 and TA1537 strains of Salmonella (Ames test) with and without metabolic activation, and also in mouse lymphoma L5178Y cells by means of micronucleus and comet assays. In microbes the drug was tested at concentrations up to 500 μg/plate and in mouse lymphoma cells up to 2,000 μg/ml.
RESULTS
In both test-systems in all experiments completely negative results were obtained although FS-1 was tested at maximum tolerated doses.
CONCLUSIONS
The drug is not genotoxic. This is advantageous because many antibacterial/antiviral drugs possess such activity.
Topics: Animals; Cell Line, Tumor; Iodophors; Lymphoma; Mice; Microsomes; Mutagenicity Tests; Polysaccharides; Salmonella
PubMed: 26011355
DOI: No ID Found -
Obesity (Silver Spring, Md.) Jan 2007Prostaglandin (PG)E2 is a lipid mediator implicated in inflammatory diseases and in the regulation of lipolysis and adipocyte differentiation. This work was, thus,...
OBJECTIVE
Prostaglandin (PG)E2 is a lipid mediator implicated in inflammatory diseases and in the regulation of lipolysis and adipocyte differentiation. This work was, thus, undertaken to study the regulation of the various PGE2 synthases (PGESs) in obesity.
RESEARCH METHODS AND PROCEDURES
C57Bl/6 mice were subjected to a high-fat or regular diet for 12 weeks. The levels of PGE2 in white adipose tissue (WAT) of lean and obese mice were quantified by liquid chromatography-mass spectrometry, and the change in expression of the three major PGES caused by diet-induced obesity was characterized by Western blotting. Human preadipocytes and 3T3-L1 cells were used to assess the expression of microsomal prostaglandin E2 synthase-1 (mPGES-1) during adipogenesis.
RESULTS
mPGES-1, mPGES-2, and cytosolic PGES proteins were all detected in WAT of lean animals. mPGES-1 was expressed at higher levels in WAT than in any other tissues examined and was more abundant (3- to 4-fold) in epididymal (visceral) compared with inguinal (subcutaneous) WAT. Expression of mPGES-1 was also detected in undifferentiated and differentiated 3T3-L1 cells and in human primary subcutaneous preadipocytes at all stages of adipogenesis. The mPGES-1 protein was substantially down-regulated in epididymal and inguinal WAT of obese mice, whereas mPGES-2 and cytosolic PGES remained relatively stable. Concordantly, the PGE2 levels in obese inguinal WAT were significantly lower than those of lean animals.
DISCUSSION
These data suggest that mPGES-1 is the major form of PGESs contributing to the synthesis of PGE2 in WAT and that its down-regulation might be involved in the alterations of lipolysis and adipogenesis associated with obesity.
Topics: 3T3-L1 Cells; Adipogenesis; Adipose Tissue; Animals; Blotting, Western; Down-Regulation; Humans; Intramolecular Oxidoreductases; Male; Mice; Mice, Inbred C57BL; Microsomes; Prostaglandin-E Synthases; Random Allocation
PubMed: 17228032
DOI: 10.1038/oby.2007.514 -
The Journal of Biological Chemistry Jan 1969
The synthesis and secretion of gamma-globulin by lymph node cells. V. The dissociation of the microsome-antibody complex with enzymatically and chemically altered gamma-globulins.
Topics: Alkylation; Amides; Animals; Antigen-Antibody Reactions; Binding Sites; Carbon Isotopes; Chemical Phenomena; Chemistry; Chromatography, Gel; Dinitrophenols; Guinea Pigs; Haptens; Iodine Isotopes; Lymph Nodes; Microsomes; Oxidation-Reduction; Papain; Pepsin A; Peptides; Periodic Acid; Protein Hydrolysates; Rabbits; Solubility; Sulfides; Time Factors; gamma-Globulins
PubMed: 4180044
DOI: No ID Found -
Proceedings of the National Academy of... May 1977Poly(A)-containing mRNA isolated from the islets of Langerhans obtained from two species of fish, angler fish (Lophius americanus) and sea raven (Hemitripterus...
Poly(A)-containing mRNA isolated from the islets of Langerhans obtained from two species of fish, angler fish (Lophius americanus) and sea raven (Hemitripterus americanus), stimulated protein synthesis 16-fold in a wheat germ cell-free system. Characterization of the translation products by polyacrylamide gel electrophoresis in sodium dodecyl sulfate showed a major polypeptide weighing 11,500 daltons that was specifically precipitated by an antibody against angler fish insulin. Partial sequence analysis of the amino terminal revealed that this polypeptide is preproinsulin, in which the amino terminus of proinsulin is preceded by either 23 (angler fish) or 25 (sea raven) amino acid residues. Translation of fish islet mRNA in a wheat germ cell-free system in the presence of dog pancreas microsomal membranes led to the correct cleavage of the nascent preproinsulin, resulting in the synthesis of authentic fish proinsulin, as verified by partial sequence analysis. Moreover, the synthesized fish proinsulin was segregated, presumably into the luminal space of the dog pancreas microsomal vesicles, because it was found to be resistant to proteolysis by added trypsin and chymotrypsin. Our data thus suggest that the mechanisms and information for the transfer of secretory proteins across the microsomal membrane are highly conserved during evolution.
Topics: Animals; Cell-Free System; Dogs; Fishes; Islets of Langerhans; Membranes; Microsomes; Molecular Weight; Pancreas; Poly A; Proinsulin; Protein Precursors; RNA, Messenger; Species Specificity; Triticum
PubMed: 325565
DOI: 10.1073/pnas.74.5.2059 -
The Journal of Biological Chemistry Dec 1986The cytochrome P-450-mediated reactions of the synthetic stilbene estrogen (E)-diethylstilbestrol (DES) and of 2-hydroxyestradiol have been investigated in vitro....
The cytochrome P-450-mediated reactions of the synthetic stilbene estrogen (E)-diethylstilbestrol (DES) and of 2-hydroxyestradiol have been investigated in vitro. Depending on the cofactor used, microsomal enzymes catalyzed reductions and/or oxidations of the estrogens: Phenobarbital-induced rat liver microsomes catalyzed the oxidation of DES to 4',4"-diethylstilbestrol quinone (DES quinone) with cumene hydroperoxide as cofactor. The quinone was unstable and spontaneously rearranged to (Z,Z)-dienestrol. DES quinone was reduced to a mixture of E- and Z-isomers of DES by NADPH catalyzed by purified cytochrome P-450 reductase. After rearrangement of the quinone to (Z,Z)-dienestrol, reduction reactions did not proceed. Rat liver microsomes and NADPH catalyzed the conversion of DES to (Z,Z)-dienestrol and (Z)-DES, but DES quinone could not be detected. The reactions described provide direct evidence for microsome-mediated redox cycling of estrogens. Although DES quinone could not be detected in the incubation of DES, microsomes, and NADPH as cofactor, the intermediacy of the quinone is demonstrated by the formation of (Z,Z)-dienestrol, the marker product for oxidation. The quinone could not be detected because it was rapidly reduced to DES and its Z-isomer. Microsome-mediated redox cycling between 2-hydroxyestradiol and the corresponding quinone was also demonstrated. Using cumene hydroperoxide as cofactor, the oxidation to the quinone was favored, while with NADPH as cofactor the reduction to 2-hydroxyestradiol was preferred. It is postulated that microsome-mediated redox cycling of estrogens plays a role in hormonal carcinogenesis.
Topics: Animals; Cytochrome P-450 Enzyme System; Diethylstilbestrol; Kinetics; Microsomes, Liver; Oxidation-Reduction; Rats; Spectrophotometry, Ultraviolet
PubMed: 3782146
DOI: No ID Found -
The Journal of Experimental Medicine May 1985Children with autoimmune chronic active hepatitis may have high titers of antibodies detected by immunofluorescence staining of hepatocytes and tubular cells in rat...
Children with autoimmune chronic active hepatitis may have high titers of antibodies detected by immunofluorescence staining of hepatocytes and tubular cells in rat liver and kidney sections, respectively. These antibodies are directed against antigens contained in microsomal fractions prepared from these two organs. We have found that sera from these patients recognized a 50,000 mol wt protein present in higher concentration in smooth microsome subfractions compared with rough microsome subfractions. This protein is an integral membrane protein and is not glycosylated. It is exposed on the cytoplasmic face of the endoplasmic reticulum and is rather resistant to proteolysis with proteinase K. Since patients with liver disease of different etiology and similar severity of cell lysis do not give rise to liver-kidney microsome antibody (LKMA), lysis of hepatocytes is apparently not a sufficient condition for their development.
Topics: Animals; Antigens; Autoantibodies; Autoantigens; Child; Endoplasmic Reticulum; Female; Humans; Kidney; Membrane Proteins; Microsomes; Microsomes, Liver; Molecular Weight; Rats; Rats, Inbred Strains; Subcellular Fractions
PubMed: 3989471
DOI: 10.1084/jem.161.5.1231 -
Neuroscience Jul 1992Immunofluorescence and immunogold labeling, together with sucrose gradient separation and Western blot analysis of microsomal subfractions, were employed in parallel to...
Immunofluorescence and immunogold labeling, together with sucrose gradient separation and Western blot analysis of microsomal subfractions, were employed in parallel to probe the endoplasmic reticulum in the cell body and dendrites of rat cerebellar Purkinje neurons. Two markers, previously investigated in non-nerve cells, the membrane protein p91 (calnexin) and the lumenal protein BiP, were found to be highly expressed and widely distributed to the various endoplasmic reticulum sections of Purkinje neurons, from the cell body to dendrites and dendritic spines. An antibody (denominated anti-rough-surfaced endoplasmic reticulum), which recognized two membrane proteins, p14 and p40, revealed a similar immunogold labeling pattern. However, centrifugation results consistent with a widespread distribution were obtained for p14 only, while p40 was concentrated in the rough microsome-enriched subfractions. The areas enriched in the inositol 1,4,5-triphosphate receptor and thus presumably specialized in Ca2+ transport (stacks of multiple smooth-surfaced cisternae; the dendritic spine apparatus) also exhibited labeling for BiP and p91, and were positive for the anti-rough-surfaced endoplasmic reticulum antibody (presumably via the p14 antigen). Additional antibodies, that yielded inadequate immunocytochemical signals, were employed only by Western blotting of the microsomal subfractions, while the ryanodine receptor was studied by specific binding. The latter receptor and the Ca2+ ATPase, known in other species to be concentrated in Purkinje neurons, exhibited bimodal distributions with a peak in the light and another in the heavy subfractions. A similar distribution was also observed with another lumenal protein, protein disulfide isomerase. Taken as a whole, the results that we have obtained suggest the existence in the endoplasmic reticulum of Purkinje neurons of two levels of organization; the first identified by widespread, probably general markers (BiP, p91, possibly p14 and others), the second by specialization markers, such as the inositol 1,4,5-triphosphate receptor and, possibly, p40, which appear restricted to areas where specific functions appear to be localized.
Topics: Animals; Blotting, Western; Calcium; Calcium Channels; Dendrites; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Fluorescent Antibody Technique; Immunohistochemistry; Male; Microsomes; Purkinje Cells; Rats; Ryanodine; Subcellular Fractions
PubMed: 1331857
DOI: 10.1016/0306-4522(92)90111-e -
The Journal of Biophysical and... Nov 1956The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its...
The pancreatic exocrine cell of the guinea pig has a voluminous endoplasmic reticulum distinguished by extensive association with small, dense particles, and by its orderly disposition in the basal region of the cell. In addition to the small, ( approximately 15 mmicro), dense particles attached to the limiting membrane of the endoplasmic reticulum, numerous particles of similar appearance are found freely scattered in the cytoplasmic matrix. The various cell structures of pancreatic exocrine cells can be satisfactorily identified in pancreatic homogenates. The microsome fraction consists primarily of spherical vesicles (80 to 300 mmicro), limited by a thin membrane (7 mmicro) which bears small ( approximately 15 mmicro) dense particles attached on its outer surface. The content of the microsomal vesicles is usually of high density. Pancreatic microsomes derive by extensive fragmentation mainly from the rough surfaced parts of the endoplasmic reticula of exocrine cells. A few damaged mitochondria and certain dense granules ( approximately 150 mmicro) originating probably from islet cells, contaminate the microsome fraction. Pancreatic microsomes contain RNA, protein, and a relatively small amount of phospholipide and hemochromogen. They do not have DPNH-cytochrome c reductase activity. In six experiments the RNA/protein N ratios were found grouped around two different means, namely 0.6 and 1.3. Pancreatic microsomes are more labile than liver microsomes but react in a similar way to RN-ase-(loss of the particulate component and RNA), and deoxycholate treatment (loss of the membranous component and of phospholipide, hemochromogen, and most of the protein). Postmicrosomal fractions consisting primarly of small ( approximately 15 mmicro), dense particles of ribonucleoprotein (RNA/protein N ratio = 1 to 2) were obtained by further centrifugation of the microsomal supernatant. The small nucleoprotein particles of these fractions are frequently found associated in chains or clusters.
Topics: Animals; Cytoplasm; Electrons; Endoplasmic Reticulum; Guinea Pigs; Histological Techniques; Microscopy; Microscopy, Electron; Microsomes; Microsomes, Liver; Pancreas; Phospholipids; Proteins; RNA
PubMed: 13398437
DOI: 10.1083/jcb.2.6.671 -
Journal of Biochemistry Mar 1984Dependence on the salt concentration of the activity of microsome-bound arylsulfatase C [EC 3.1.6.1] from rat liver was examined. The activity increased with increasing...
Dependence on the salt concentration of the activity of microsome-bound arylsulfatase C [EC 3.1.6.1] from rat liver was examined. The activity increased with increasing salt concentration in the reaction medium in the whole pH range tested. This effect can be explained by the dependence of the reaction rate on the surface pH and the surface concentration of the ionic substrate. The dependence on salt concentration of the activity of the microsome-bound arylsulfatase C and the pH-dependences of Vmax and Km of the enzyme were used for the estimation of pH at the microsomal surface. The two values of the surface pH (surface potential) and the salt concentration were applied to the Gouy-Chapman equation. The value of -0.39 +/- 0.08 X 10(-3) elementary charge/A2 was obtained as the surface charge density in the vicinity of the microsome-bound arylsulfatase C. This was smaller than the over-all value for microsomes (-1.08 +/- 0.04 X 10(-3) elementary charge/A2; Masamoto, K. (1982) J. Biochem. 92, 365-371). This suggests that the anion concentration in the vicinity of the enzyme on microsomes is lower than that in the bulk aqueous phase and is higher than the average value at the microsomal surface when the salt concentration is low.
Topics: Animals; Arylsulfatases; Binding Sites; Electrochemistry; Hydrogen-Ion Concentration; Microsomes, Liver; Models, Chemical; Osmolar Concentration; Rats; Steryl-Sulfatase; Substrate Specificity; Sulfatases; Surface Properties
PubMed: 6586719
DOI: 10.1093/oxfordjournals.jbchem.a134661