The effects of 7-ethyl-8-methylf1avin (7-Et) and 7-methyl-8-ethyl-flavin (8-Et) on rat hepatic monoamine oxidase (MAO), brain MAO activity and 5-hydroxytryptamine (5-HT or serotonin) in rat brain were investigated. In the study of hepatic MAO activity, kynur-amine a nonphysiological substrate for both A and B type MAO, was used for a spectro-photometric method, and [14C]-labeled amines were also used for a radiometric procedure for camparison with MAO activity determined by the spectrophotometric method. The rate of change in MAO activity of hepatic mitochondria from rats receiving Rb-def and 7-Et and 8-Et flavin showed the activity was severely reduced during 8 weeks. Rapid reduction of enzyme activity (50% in def-group, 35% in 7-Et group and 8% 8-Et flavin group) was observed at the end of 2 weeks. The enzyme activity lasted with slow decre-ment of enzyme level from 4 weeks to the end of 8 weeks as low as 16% in def, 18% in 7-Et and 3% in 8-Et flavin group. The trend of decrement of MAO activity when kynura-mine was used as a substrate appears to be similar with the small variation of MAO activity when [14C]-labelled tyramine, dopamine, serotonin and tryptamine respectively were used as substrate. The rate of decay of brain mitochondrial MAO activity in rats receiving each respective f1avin was not rapid and severely depressed as the MAO activity we have found in liver mitochondrial MAO of rats during the 8 week experimental time, but a similar tendency of decay of MAO in each group was observed. The potent inhibitory effect of 8-Et on brain MAO was confirmed by the study of the simultaneous measure-ment of MAO activity in each experimental group. when the reduction Of brain MAO activity in rats receiving 8-Et after 6 weeks was approximately 80% of normal and in the same rats the concentration of brain 5-HT showed a 60% increment of that of the normal mts. During the experimental period there is no absolute parallelism between the MAO inhibition and 5-HT increase. However when the reduction of MAO activity reached 80% of normal value, the concentration of 5-HT increased dramatically as much as 60% of normal value. The results so far suggest clearly that 8-Et produces a much more potent inhibitory effect on the hepatic MAO a s well as brain MAO in rats. Therefore our present and previous results suggest that 7-Et and 8-Et flavin should bind itself to hepatic, brain MAO apoenzyme in the condition of total absence of riboflavin in these animals, and the holenzyme is catalytically inactive.
Animal ; Brain/enzymology* ; Brain/metabolism ; Comparative Study ; Male ; Mitochondria/enzymology* ; Mitochondria, Liver/enzymology* ; Monoamine Oxidase/metabolism* ; Rats ; Serotonin/metabolism*

Methylation catalyzed by methyltransferases is a major metabolic pathway for an inactivation of some catecholamines, niacinamide as well as aliphatic sulfhydryl drugs and toxic hydrogen sulfides. To investigate the effects of obstructive jaundice in an animal model, common bile duct ligation (CBDL) was performed in the rat and enzyme activities of S-adenosyl-L-methionine-dependent arylamine N-methyltransferase and thiol methyltransferase were examined in liver cell fractions and serum for a period of 42 d after CBDL. Both mitochondrial and microsomal arylamine N-methyltransferase showed significant increases in their activities between the 1st through the 7th day (P < or = 0.05 to 0.001), and between the 1st through the 28th day (P X or = 0.01 to 0.001) post-ligation, although the cytosolic arylamine N-methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. The mitochondrial as well as microsomal thiol methyltransferase showed significant increases in their activities between the 1st through the 28th day (P < or = 0.05 to 0.01 and P < or = 0.01 to 0.001, respectively) post-ligation, although the cytosolic thiol methyltransferase activity did not show a significant change compared to the activities from the sham-operated control. Arylamine N-methyltransferase and thiol methyltransferase in the serum from cholestatic rats also showed significant increases in their activities between the 1st through 28th day (P < or = 0.01 to 0.001), and between the 0.5th through the 42nd day (P < or = 0.05 to 0.001) post-ligation compared to the sham-operated control, respectively. Enzyme kinetic parameters (Km and Vmax) of hepatic membrane-bound arylamine N-methyltransferase and thiol methyltransferase were analyzed with the preparation from the 7th day post-ligation, using tryptamine or 4-chlorothiophenol as substrates and S-Adenosyl-L-[methyl-3H]methionine as co-substrate. The results indicate that although the Km values were about the same as the sham-operated control, the Vmax values of both enzymes increased significantly (P < or = 0.01 and 0.001, respectively). These results suggest that the biosynthesis of arylamine N-methyltransferase and thiol methyltransferase have been induced in response to obstructive jaundice.
Animal ; Bile Ducts/surgery ; Cholestasis/*enzymology ; Ligation ; Liver/*enzymology ; Methyltransferases/blood/*metabolism ; Microsomes, Liver/enzymology ; Mitochondria, Liver/enzymology ; Rats ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVETo investigate the effects of seawater immersion on the function of myocardium and hepatocyte mitochondria in experimental hemorrhagic shock rats.

METHODSTwenty-four male Wistar rats were divided into three groups (n=8 in each group): control group, HSL group (hemorrhagic shock group on land) and HSS group (hemorrhagic shock group in seawater). The hemodynamic parameters, activities of H(+)-ATPase (adenosinetriphosphatase), succinate dehydrogenase (SDH) and Ca(2+)-Mg(2+)-ATPase, the calcium contents in myocardium and hepatocyte mitochondria were measured and the changes of proton translocation across the inner mitochondrial membrane were analyzed.

RESULTSThe hemodynamic indexes and the activities of H+-ATPase, SDH, Ca(2+)-Mg(2+)-ATPase in HSS group were significantly lower than those in control group and HSL group (P<0.05). In HSS group the calcium levels in tissue and mitochondria of myocardium and hepatocyte were elevated significantly compared with control group and HSL group (P<0.05). There was no significant difference in proton translocation among three groups.

CONCLUSIONSThis investigation demonstrates that seawater immersion can aggravate the conditions of hemorrhagic shock rats.

Animals ; Calcium ; metabolism ; Immersion ; Male ; Mitochondria, Heart ; enzymology ; Mitochondria, Liver ; enzymology ; Proton-Translocating ATPases ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Seawater ; Shock, Hemorrhagic ; enzymology ; physiopathology

OBJECTIVETo investigate the changes of proton transportation across the inner mitochondrial membrane (IMM) and H(+)-ATPase of hepatocytes in endotoxic shock rats.

METHODSEndotoxin from E. Coil of 5.0 mg/kg or saline of 1 ml/kg was injected into the femoral vein. The rats were sacrificed pre-injection and 1, 3, 5, 8 hours after injection, and plasma and liver tissue samples were collected respectively. The liver tissue samples were used for preparation of mitochondria and submitochondrial particles (SMPs). The proton-translocation of SMPs and H(+)-ATPase, phospholipase A(2) (PLA(2)) activities and malondialdehyde (MDA) content, membrane fluidities of different level of mitochondria membrane and plasma MDA content were assayed.

RESULTS(1) Five hours after E. Coli. O111B4 injection, the maximum fluorescence quenching ACMA after adding ATP, nicotinamide adenin dinucleoacid hydrogen (NADH), and the succinate were significantly decreased (P<0.05). The time of maximum fluorescent quenching and the half time of fluorescent quenching were significantly prolonged (P<0.01), especially when NADH was used as a substrate. (2) The mitochondrial H(+)-ATPase activity was significantly increased at early stage of endotoxic shock (P<0.05), and significantly decreased at late stage of endotoxic shock (P<0.01). (3) The mitochondrial membrane bound PLA(2) activity, plasmal and mitochondrial MDA content were significantly increased and succinate dehydrogenase (SDH) activity of mitochondria decreased markedly in endotoxic shock rats (P<0.05). (4) The mitochondrial membrane fluidity of different lipid regions was decreased, especially in the head of phospholipid.

CONCLUSIONSProton transportation across IMM and mitochondrial H(+)-ATPase activity are significantly decreased in endotoxic shock.

Animals ; Microscopy, Electron ; Mitochondria, Liver ; metabolism ; Proton-Translocating ATPases ; metabolism ; Rats ; Rats, Wistar ; Shock, Septic ; enzymology

OBJECTIVETo explore the mitochondrial toxicities induced by zidovudine (AZT) and adefovir dipivoxil (ADV) antiviral drugs using a rat model system.

METHODSTwelve healthy Sprague-Dawley rats were randomly divided into three equal groups and treated by oral gavage with zidovudine (125 mg/kg/day), adefovir (40 mg/kg/day), or saline (equal volume) for 28 days. The rats' body weights were measured once a week, and blood was collected every two weeks for blood and biochemical tests. All animals were sacrificed at the end of treatment, and liver, kidney, skeletal muscle, and cardiac muscle were collected by necropsy. Mitochondria were isolated from the respective tissue samples, and the activities of respiratory chain complexes were measured. DNA was purified from each sample and the mitochondrial DNA (mtDNA) content was monitored by quantitative real time PCR. Mitochondrial morphology was analyzed under electron microscope.

RESULTSNo significant adverse effects, including body weight loss, abnormal blood or biochemistry, were observed in rats treated with AZT or ADV. The activities of mitochondrial cytochrome c oxidase in liver and cardiac muscle were slightly decreased in rats treated with AZT (liver: 9.44+/-3.09 vs. 17.8+/-12.38, P?=?0.21; cardiac muscle: 32.74+/-5.52 vs. 24.74+/-20.59, P?=?0.28; kidney: 4.42+/-1.53 vs. 14.45+/-13.75, P?=?0.18; skeletal muscle: 33.75+/-8.74 vs. 40.04+/-2.49, P?=?0.45). The mtDNA content was significantly decreased in cardiac muscle of AZT-treated rats (cardiac muscle: 0.15+/-0.13 vs. 0.32+/-0.42, P?=?0.85). The morphology of mitochondria in liver, kidney, skeletal muscle, and cardiac muscle was significantly altered in the AZT-treated rats and included disappearance of the outer membrane, severely damaged structure, and swollen or completely absent cristae. No obvious effects were noted in the ADV- or saline-treated rats.

CONCLUSIONSignificant adverse effects related to mitochondrial toxicity were observed in rats treated with AZT. The slightly decreased mtDNA content in ADV-treated rats may suggest that this antiviral drug can also cause mitochondrial toxic effects.

Adenine ; adverse effects ; analogs & derivatives ; Animals ; DNA, Mitochondrial ; drug effects ; Electron Transport Complex IV ; metabolism ; Female ; Kidney ; enzymology ; Liver ; enzymology ; Mitochondria ; drug effects ; metabolism ; Mitochondria, Heart ; drug effects ; Mitochondria, Liver ; drug effects ; Mitochondria, Muscle ; drug effects ; Muscle, Skeletal ; enzymology ; Myocardium ; enzymology ; Organophosphonates ; adverse effects ; Rats ; Rats, Sprague-Dawley ; Zidovudine ; adverse effects

OBJECTIVETo investigate the intervention effect of aqueous fractions from Boschniakia rossica (BRAF) on hepatic oxidative stress in mice with liver injury induced by carbon tetrachloride (CCl4).

METHODThe experimental mice were randomly assigned into the normal control group, the model group, the silymarin (positive control) group, as well as high and low dose BRAF groups. Mice were treated intragastrically with silymarin or BRAF once every day for 7 days. At the end of the experiment, CCl4 was injected intraperitoneally into the mice to establish the acute liver injury model. The pathological changes was detected with hematoxylin and eosin (HE) staining, and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), superoxide dismutase (SOD) , catalase (CAT), glutathione peroxidase (GPx), Na+ -K+ -ATPase, Ca2+ -Mg2+ -ATPase, and the contents of reduced glutathione (GSH) and malondialdehyde (MDA) were detected by the colorimetric method.

RESULTBRAF significantly reduced ALT, AST and ALP activities in serum, alleviated hepatic injury induced by CCl4, increased SOD, CAT, GPx and GSH levels in liver, and SOD, Na + -K + -ATPase and Ca2+ -Mg2 + -ATPase activities in liver mitochondria, and decreased the MDA content in liver and liver mitochondria.

CONCLUSIONBRAF reduces hepatic oxidative stress in mice with acute liver injury induced by CCl4, thereby showing the protective effect on mice with acute liver injury induced by CCl4.

Animals ; Carbon Tetrachloride ; toxicity ; Chemical and Drug Induced Liver Injury ; enzymology ; metabolism ; pathology ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Liver ; drug effects ; enzymology ; metabolism ; pathology ; Male ; Mice ; Mitochondria ; drug effects ; metabolism ; Orobanchaceae ; chemistry ; Oxidative Stress ; drug effects ; Solubility ; Water ; chemistry

The activity of Mg++-dependent, Ca++-activated adenosine triphosphatase (Ca-ATPase) of rat liver mitochondria was studied at varying medium compositions, pH and temperatures. The enzyme system was characteristically sensitive to Ca++ concentration with a KmCa of approximately 0.06 mM. The optimal concentration of Mg was about l mM, above which the enzyme activity was progressively inhibited. The inhibitory effect of high Mg++ concentrations appeared to be due to the alteration of the Mg++/ATP ratio. Variations in the Mg++/ATP ratio affected Vmax but not the KmATP. The pH optimum for enzyme activity increased as the incubation temperature decreased, but the optimal OH-/H+ ratio of the medium was constant at around 0.1, regardless of temperature. The activity of the enzyme was not affected by La# (0.01-1 mM) and Ruthenium red (2.5-10.0 microM). These results indicate that 1) the enzymatic characteristics of the Ca-ATPase system in the rat liver mitochondria is typical of those from other tissue preparations, 2) the enzyme system maintains the most effective catalytic conformation at a fixed level of OH-/H+ ratio of 0.1 when the temperature changes, and 3) the enzyme system may not play a role in the physiological transport of Ca++ in mitochondria.
Animal ; Ca(2+)-Transporting ATPase/metabolism* ; Calcium/pharmacology ; Enzyme Activation/drug effects ; Female ; Hydrogen-Ion Concentration ; Magnesium/pharmacology ; Male ; Mitochondria, Liver/enzymology* ; Rats ; Temperature

OBJECTIVETo study the effects of Angelica sinensis Polysaccharides (ASP) on the hepatic drug metabolism enzymes activities in normal mice and those prednisolone (PSL)-induced liver injury.

METHODThe activities of phase II enzymes (GSH-related enzymes) and cytochrome P450 enzymes were measured by biochemical method.

RESULTASP increased the activities of glutathione S-transferase in liver microsomes and mitochondria. The cytochrome P450 content, NADPH-cytochrome c reductase, aminopyrine N-demethylase, and aniline hydroxylase activities in liver microsomes were also increased. PSL significantly increased serum ALT levels, and decreased the liver mitochondrial glutathione content. At the same time, other enzymes activities were all increased. When mice were treated with ASP 2.0 g.kg-1, the PSL-induced changes on cytochrome P450 enzymes, glutathione S-transferase, and GSH content were restored.

CONCLUSIONASP can modulate the activities of drug metabolism enzymes.

Aminopyrine N-Demethylase ; metabolism ; Angelica sinensis ; chemistry ; Aniline Hydroxylase ; metabolism ; Animals ; Chemical and Drug Induced Liver Injury ; enzymology ; etiology ; Cytochrome P-450 Enzyme System ; metabolism ; Glutathione Transferase ; metabolism ; Male ; Mice ; Microsomes, Liver ; enzymology ; Mitochondria, Liver ; enzymology ; NADPH-Ferrihemoprotein Reductase ; metabolism ; Plants, Medicinal ; chemistry ; Polysaccharides ; isolation & purification ; pharmacology ; Prednisolone

Primary rat hepatocytes were cultured using different in vitro models and the enzyme leakage, albumin secretion, and cytochrome P450 1A (CYP 1A) activity were observed. The results showed that the level of LDH was decreased over time in culture. However, on day 5, LDH showed a significant increase in monolayer culture (MC) while after day 8 no LDH was detectable in sandwich culture (SC). The levels of AST and ALT did not change significantly over the investigated time. The CYP 1A activity was gradually decreased in a time-dependent manner in MC and SC. The decline of CYP 1A was faster in MC than in SC. This effect was partially reversed by using cytochrome P450 (CYP450) inducer such as Omeprazol and 3-methylcholanthrene (3-MC) and the CYP 1A induction was always higher in MC than in SC. In bioreactor basic CYP 1A activity was preserved over 2 weeks and the highest albumin production was observed in bioreactor followed by SC and MC. Taken together, it was indicated each investigated model had its advantages and disadvantages. It was also underlined that various in vitro models may address different questions.
Albumins/*secretion ; Bioreactors ; Cell Culture Techniques/*methods ; Cell Separation ; Cytochrome P-450 CYP1A1/metabolism ; Hepatocytes/*cytology ; Hepatocytes/metabolism ; L-Lactate Dehydrogenase/metabolism ; Mitochondria, Liver/enzymology ; Mitochondrial Proteins/metabolism ; Rats, Sprague-Dawley ; Time Factors

OBJECTIVETo determine the roles of mitochondrial apoptosis and energy metabolism in hepatocytes during the pathogenic process of acute renal failure (ALF) by assessing disease-related differential activities of several key mitochondrial enzymes, including citrate synthase (CS), carnitine palmitoyltransferase-1 (CPT-1) and cytochrome c oxidase (COX).

METHODSThirty-two male Sprague Dawley rats were given D-galactosamine followed by and lipopolysaccharide (LPS) to induce acute liver failure and sacrificed after 4 (4 h group), 8 (8 h group) 12 (12 h group) and 24 hours (24 h group) of treatment. Eight unmodeled rats served as controls. Effects related to apoptosis were evaluated by pathological analysis of hepatic tissues and TUNEL staining. Ultrastructural changes in mitochondria were assessed by electron microscopy. The activity and expression of CS, CPT-1 and COX were measured.

RESULTSHepatocyte apoptosis was present in the 4 h treatment group and was increased obviously in the 8 h treatment group. Hepatocyte necrosis was first observed in the 12 h treatment group and was significantly higher in the 24 h treatment group, with inflammatory cell invasion. Ultrastructural changes in mitochondria were present in the 4 h treatment group, and the 24 h treatment group showed mitochondria with completely destroyed outer membranes, which resulted in mitochondrial collapse. Activity and protein expression of CS, CPT-1 and COX were increased in the 4 h group (vs. controls), were at their peak in the 8 h group (CS:t =1.481, P less than 0.01; CPT-1:t =2.619, P less than 0.05; COX:t =1.014, P less than 0.01) and showed a decreasing trend in the 12 h group. In addition, the activities of CS, CPT-1 and COX were enhanced at the stage of hepatocyte apoptosis, suggesting that these enzymes were involved in the initiation and development of ALF.

CONCLUSIONEnergy metabolism plays an important role in hepatocyte apoptosis during ALF.

Animals ; Apoptosis ; Carnitine O-Palmitoyltransferase ; metabolism ; Citrate (si)-Synthase ; metabolism ; Disease Models, Animal ; Electron Transport Complex IV ; metabolism ; Hepatocytes ; cytology ; enzymology ; Liver Failure, Acute ; metabolism ; pathology ; Male ; Mitochondria ; ultrastructure ; Rats ; Rats, Sprague-Dawley