The contribution of thioredoxin-2 reductase and glutathione peroxidase to H(2)O(2) detoxification of rat brain mitochondria

Brain mitochondria are not only major producers of reactive oxygen species but they also considerably contribute to the removal of toxic hydrogen peroxide by the glutathione (GSH) and thioredoxin-2 (Trx2) antioxidant systems. In this work we estimated the relative contribution of both systems and catalase to the removal of intrinsically produced hydrogen peroxide (H(2)O(2)) by rat brain mitochondria. By using the specific inhibitors auranofin and 1-chloro-2,4-dinitrobenzene (DNCB), the contribution of Trx2- and GSH-systems to reactive oxygen species (ROS) detoxification in rat brain mitochondria was determined to be 60±20% and 20±15%, respectively. Catalase contributed to a non-significant extent only, as revealed by aminotriazole inhibition. In digitonin-treated rat hippocampal homogenates inhibition of Trx2- and GSH-systems affected mitochondrial hydrogen peroxide production rates to a much higher extent than the endogenous extramitochondrial hydrogen peroxide production, pointing to a strong compartmentation of ROS metabolism. Imaging experiments of hippocampal slice cultures showed on single cell level substantial heterogeneity of hydrogen peroxide detoxification reactions. The strongest effects of inhibition of hydrogen peroxide removal by auranofin or DNCB were detected in putative interneurons and microglial cells, while pyramidal cells and astrocytes showed lower effects. Thus, our data underline the important contribution of the Trx2-system to hydrogen peroxide detoxification in rat hippocampus. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).     

Assignment of two rat dihydrofolate (DHFR) genes to chromosomes 2 and 4

A panel of rat x mouse cell hybrids was used in the chromosomal mapping of the rat dihydrofolate reductase (DHFR) gene. It was determined that the probe hybridized to gene sequences on two different chromosomes (Nos. 2 and 4), possibly representing the active gene and a pseudogene. Hybridization of the DHFR probe to DNA from a methotrexate resistant rat cell line revealed that the gene on chromosome 2 was amplified, but not the gene on chromosome 4. This result was taken to suggest that the active DHFR gene is located on rat chromosome 2 and that the sequence on chromosome 4 is a pseudogene.     

The use of rat Leydig tumor (R2C) and human hepatoma (HEPG2) cells to evaluate potential inhibitors of rat and human steroid aromatase

The efficacies of 10-propargylestr-4-ene-3,17-dione (PED), 4-hydroxyandrostenedione (4-OHA) and the imidazole broad spectrum antimycotic drugs, econazole, imazalil, miconazole and ketoconazole, to inhibit the steroid aromatase activities of rat Leydig tumor (R2C) cells and human hepatoma (HEPG2) cells have been determined. The analysis of inhibition of steroid aromatase activity of intact cells provided further insight into the potential use of such drugs to block cellular estrogen synthesis. The IC50 values for the inhibition of aromatase activity of R2C cells by econazole, imazalil, miconazole, ketoconazole, 4-OHA and PED were 4, 9, 40, 1100, 11 and 10 nM, respectively. These drugs also inhibited the steroid aromatase activity of HEPG2 cells with corresponding IC50 values of 13, 27, 20, 15000, 2 and 2 nM, respectively; these findings were suggestive that the steroid aromatase of rat has many similarities to the human enzyme in its interaction with putative inhibitory compounds. Importantly, however, ketoconazole inhibited the rat aromatase more effectively than it did the human enzyme, while PED and 4-OHA were less effective inhibitors of the rat enzyme compared to that of human. These findings indicate differences in the potencies of various drugs to inhibit estrogen biosynthesis in human and rat cells. These may relate to differences in the two aromatase systems and/or differences in the stability of the drugs in the human hepatoma and rat Leydig tumor cells.     

Hemoproteins affect H(2)O(2) removal from rat tissues

The capacity of rat liver homogenates and mitochondria to remove H(2)O(2) was determined by comparing their ability to slow fluorescence generated by a H(2)O(2) 'detector' with that of desferrioxamine solutions. H(2)O(2) was produced by glucose oxidase-catalysed glucose oxidation. The capacity to remove H(2)O(2) was expressed as equivalent concentration of desferrioxamine. The method showed changes in the capacity of H(2)O(2) removal after treatment with ter-butylhydroperoxide or glutathione. The H(2)O(2) removal capacity of homogenates and mitochondria from rat liver, heart, and skeletal muscle was compared with their overall antioxidant capacity. For homogenates, the order of both antioxidant and H(2)O(2) removal capacities was liver>heart>muscle. For mitochondria, the order of the antioxidant capacities mirrored that of the homogenates, while the order of the H(2)O(2) removal capacities was heart>muscle>liver. Because H(2)O(2) removal is not only due to H(2)O(2)-metabolizing enzymes, but also to hemoproteins that convert H(2)O(2) into more reactive radicals via Fenton reaction, the higher concentration of cytochromes in mitochondria of cardiac and skeletal muscles can explain the above discrepancy. A higher H(2)O(2) removal capacity was found to be associated with a higher rate of H(2)O(2) release by mitochondria, indicating that the order of H(2)O(2) release rate mirrors that of H(2)O(2) production rate. We suggest that the different capacities of the mitochondria from the three tissues to produce reactive oxygen species are due to differences in the concentration of respiratory mitochondrial chain components in the reduced form.     

Cellular responses to H(2)O(2) and bleomycin-induced oxidative stress in L6C5 rat myoblasts

In muscle cells, reactive oxygen species (ROS) are continually generated. It is believed that these molecules have a well-established role as physiological modulators of skeletal muscle functions, ranging from development to metabolism and from blood flow to contractile functions. Moreover, ROS may contribute to the development of muscle fatigue, inflammation, and degeneration, and may be implicated in many muscle diseases. The aim of the present study was to verify the role of short or prolonged exposure to oxidative stress, generated by different concentrations of H(2)O(2), on growth, chromosomal aberrations, and apoptosis induced in cultured L6C5 rat muscle cells used as model for myoblasts. Our results indicate that, in L6C5 cells, reactive oxygen intermediates (ROI) can activate distinct cell pathways leading to cell growth induction and development of resistant phenotype, or to chromosomal aberrations, cell cycle arrest, or cell death. The positive vs. negative effects of H(2)O(2)-altered redox potential in myoblasts are strictly related to the intensity of oxidative stress, likely depending on the types and number of cellular targets involved. Among these, DNA molecules appear to be very sensitive to breakage by H(2)O(2), although DNA damage is not directly responsible for ROI-induced apoptosis in L6C5 rat myoblasts.     

Chromosomes and DNA-replication of rat kangaroo cells (PtK2)

The karyotype, chromosomal measurements, and the time course of DNA replication during the S-phase were determined in metaphase chromosomes of non-synchronized monolayer cultures of PtK₂ cells (CCL 56) derived from Potorous tridactylis. The karotype was the same as originally determined for this cell line. Chromosomal measurements differed from data for primary bone marrow cells of this species published by Shaw and Krooth. PtK₂ cells and chromosomes showed maximal incorporation of tritiated thymidine (³H-TdR) halfway through the S-phase. Chromosome Y₁ showed a second peak of ³H-TdR-incorporation at the end of the S-phase in addition to the peak halfway through S. Comparison of grain densities for chromosomal arms showed late replication of the short arms of chromosomes 1, 3, and X. The time course of incorporation of ³H-TdR was changed when cells were treated for 1 h with fluorodeoxyuridine (FUdR) prior to the ³H-TdR-pulse. FUdR-treated cells showed maximum incorporation of ³H-TdR immediately after the beginning of the S-phase, which was followed by a second peak halfway through the S-phase. This indicated that ³H-TdR-incorporation was partially synchronized by treatment of cells with FUdR. Total radioactivity of FUdR-treated cells had increased by 77% in comparison to cells not treated with FUdR, which indicates that approximately 44% of the TdR-precursors of the latter cells may have originated from cellular precursor pools.     

Generation and characterization of a Tet-On (rtTA-M2) transgenic rat

Background  

The tetracycline-inducible gene regulation system is a powerful tool that allows temporal and dose-dependent regulation of target transgene expression in vitro and in vivo. Several tetracycline-inducible transgenic mouse models have been described with ubiquitous or tissue-specific expression of tetracycline-transactivator (tTA), reverse tetracycline-transactivator (rtTA) or Tet repressor (TetR). Here we describe a Tet-On transgenic rat that ubiquitously expresses rtTA-M2 driven by the murine ROSA 26 promoter.     

Contractile responses to prostacyclin (PGI2) of isolated human saphenous and rat venous tissue.

Prostacyclin (PGI2), in a wide concentration range, produced neither contraction nor relaxation of isolated human saphenous vein. Isolated portal veins and vena cava from normal and spontaneously hypertensive rats (SHR) responded only with an increase in contractile tension when exposed to PGI2. This constrictor effect was absent in a calcium-free buffer. PGI2 failed to relax KCI contracted vena cava. The constrictor effect of PGI2 on portal vein was attenuated in a glucose-free, oxygen deficient buffer. No tachyphylaxis or tolerance to the constrictor effect of PGI2 was noted. Results emphasize that PGI2 may produce differing effects on vascular smooth muscle tension depending on species and type of blood vessel studied.     

Purification and characterization of beta-galactoside (alpha 2 leads to 6)sialyltransferase from rat liver and hepatomas

Asialofetuin sialyltransferase from Triton X-100 extracts of rat liver was resolved by phosphocellulose chromatography into two fractions, designated I and II in order of elution. When previously treated with Arthrobacter ureafaciens neuraminidase, fraction I eluted at about the same position as II while no alteration occurred in II. Primary rat hepatomas contained only a single asialofetuin sialyltransferase, identical to fraction I in chromatographic behavior. Transferases I and II were purified to near homogeneity. Transferase II, as well as neuraminidase-treated I, could be sialylated auto-catalytically, indicating that the lack of sialic acid in II is not due to the lack of a sialic-acid-accepting site. Both enzymes formed an (alpha 2 leads to 6)sialylgalactoside linkage with asialo-glycoproteins of the glycosylamine-type and with lactose, and were indistinguishable immunologically. Nevertheless, the transferases exhibited different molecular weights of 37000 (I) and 43000 (II). When heated at 50 degrees C, transferase I lost half its original activity within 20 min while II was scarcely inactivated. Kinetically, transferase I showed three-times higher affinity than II for CMP-N-acetylneuraminic acid and for desialylated plasma membrane. Asialofetuin sialyltransferase was also purified from primary rat hepatoma. The purified enzyme was identical to transferase I in every respect examined. We conclude that hepatomas contain transferase I but lack transferase II.     

Regulation of TXB(2) and PGE(2) production by TGF-beta(1) in in vitro silica dust-exposed rat alveolar macrophage

We investigated the effect of transforming factor factor-beta(1) (TGF-beta(1)) on thromboxane B(2) (TXB(2)) and prostaglandin E(2) (PGE(2)) production in in vitro silica dust-exposed rat alveolar macrophages (AM). In the presence of 5 mug of anti-TGF-beta(1) antibodies, TXB(2) production decreased, but PGE(2) production increased. Addition of 2 ng of TGF-beta(1) to the culture medium potentiated TXB(2) production, but PGE(2) production apparently did not change. At 50 ng of TGF-beta(1), TXB(2) production decreased, and PGE(2) production varied. Our data suggest that in rat AM: (1) both endogenous and exogenous TGF-beta(1) regulate TXB(2) production; and (2) in the absence of endogenous TGF-beta(1) the liberation of PGE(2) increases; however, exogenous TGF-beta(1) does not have a regulatory effect on PGE(2).     

Biochemical alterations in rat fetal liver following in utero exposure to di(2-ethylhexyl)phthalate (DEHP)

Oral administration of DEHP, 1000 mg/kg body weight, to rats daily from 6 to 15 day of gestation resulted in retardation of fetal growth and increase in fetal liver weight which contained significant quantities of DEHP. The activities of mitochondrial succinate dehydrogenase, malate dehydrogenase, cytochrome c oxidase and adenosine triphosphatase were decreased in fetal liver. The data indicate that exposure of mothers to DEHP during pregnancy could adversely affect the fetal livers by interfering with bioenergetics of the cell.     

Biosynthesis of S-(2-hydroxy-2-carboxyethylthio)-L-cysteine (3-mercaptolactate-cysteine disulfide) by the rat heart

Incubation of 3-mercaptopyruvate with rat heart homogenate resulted in the formation of S-(2-hydroxy-2-carboxy-ethylthio)-L-cysteine (HCETC, 3-mercaptolactate-cysteine disulfide), L-cysteine and 3-mercaptolactate with the concomitant decrease in glutamate and aspartate. These results indicate that a part of 3-mercaptopyruvate was converted to L-cysteine by transamination, a part was reduced to 3-mercaptolactate, and HCETC was formed from these two products. Another peak which corresponds to L-cysteine-glutathione disulfide on amino acid analysis was also produced during the incubation.     

Signaling pathways leading to prostaglandin E(2) production by rat cerebral frontal cortex

In this paper, we have determined the effect of both muscarinic acetylcholine receptor (mAChR) and exogenous prostaglandin E(2) (PGE(2)) on PGE(2) production and cyclooxygenases (COX) mRNA gene expression on rat cerebral frontal cortex. Carbachol and PGE(2) increase endogenous PGE(2) production and the COX-1 mRNA levels by activation of PLA(2)s. The COX-1 and COX-2 activity participated in the production of PGE(2) triggered by exogenous PGE(2). While in carbachol-PGE(2) only COX-1 activity is affected. The specific inhibition of PGE(2) receptor was able to impair the increase of endogenous PGE(2) production triggered by both carbachol and exogenous PGE(2). These results suggest that carbachol-activation mAChR increased PGE(2) production that in turn interacting with its own receptor triggers an additional production of PGE(2). Both mechanisms appear to occur by using PLA(2) signaling system. This data should be able to contribute to understand the involvement of PGE(2) in normal brain function and its participation in neuroinflammatory processes.     

Effect of training on H(2)O(2) release by mitochondria from rat skeletal muscle

In this study, oxygen consumption and H(2)O(2) release rate by succinate or pyruvate/malate supplemented mitochondria isolated from skeletal muscle of trained and untrained rats were investigated. The overall mitochondrial antioxidant capacity and the effect of preincubation of mitochondria with GDP, an inhibitor of uncoupling proteins UCP1 and UCP2, on both succinate-supported H(2)O(2) release and membrane potential were also determined. The results indicate that training does not affect mitochondrial oxygen consumption with both complex-I- and complex II-linked substrates. Succinate-supported H(2)O(2) release was lower in trained than in untrained rats both in State 4 and State 3. Even the antimycin A-stimulated release was lower in trained rats. When pyruvate/malate were used as substrates, H(2)O(2) release rate was lower in trained rats only in the presence of antimycin A. The increase of mitochondrial protein content (determined by the ratio between cytochrome oxidase activities in homogenates and mitochondria) in trained muscle was such that the succinate-supported H(2)O(2) release per g of tissue was not significantly different in trained and untrained rats, while that supported by pyruvate/malate was higher in trained than in untrained animals. The lack of training-induced changes in overall antioxidant capacity of mitochondria indicates that the decrease in mitochondrial H(2)O(2) release cannot be attributed to a greater capacity of mitochondria to scavenge the reactive oxygen intermediates derived from univalent O(2) reduction by respiratory chain components. In contrast, the above decrease seems to depend on the drop induced by training in mitochondrial membrane potential. These training effects are not due to an increased level of mitochondrial uncoupling protein, because in the presence of GDP the increase in both membrane potential and H(2)O(2) release was greater in untrained than in trained rats.     

Oxidative stress contributes to pulmonary hypertension in the transgenic (mRen2)27 rat

The transgenic (mRen2)27 (Ren2) rat overexpresses mouse renin in extrarenal tissues, causing increased local synthesis of ANG II, oxidative stress, and hypertension. However, little is known about the role of oxidative stress induced by the tissue renin-angiotensin system (RAS) as a contributing factor in pulmonary hypertension (PH). Using male Ren2 rats, we test the hypothesis that lung tissue RAS overexpression and resultant oxidative stress contribute to PH and pulmonary vascular remodeling. Mean arterial pressure (MAP), right ventricular systolic pressure (RVSP), and wall thickness of small pulmonary arteries (PA), as well as intrapulmonary NADPH oxidase activity and subunit protein expression and reactive oxygen species (ROS), were compared in age-matched Ren2 and Sprague-Dawley (SD) rats pretreated with the SOD/catalase mimetic tempol for 21 days. In placebo-treated Ren2 rats, MAP and RVSP, as well as intrapulmonary NADPH oxidase activity and subunits (Nox2, p22phox, and Rac-1) and ROS, were elevated compared with placebo-treated SD rats (P < 0.05). Tempol decreased RVSP (P < 0.05), but not MAP, in Ren2 rats. Tempol also reduced intrapulmonary NADPH oxidase activity, Nox2, p22phox, and Rac-1 protein expression, and ROS in Ren2 rats (P < 0.05). Compared with SD rats, the cross-sectional surface area of small PA was 38% greater (P < 0.001) and luminal surface area was 54% less (P < 0.001) in Ren2 rats. Wall surface area was reduced and luminal area was increased in tempol-treated SD and Ren2 rats compared with untreated controls (P < 0.05). Collectively, the results of this investigation support a seminal role for enhanced tissue RAS/oxidative stress as factors in development of PH and pulmonary vascular remodeling.     

Enantioselective metabolism of (R)- and (S)-4-hydroxy-2-nonenal in rat

4-Hydroxy-2-nonenal (HNE) is an endogenous product of lipid peroxidation, which is believed to play a biological role in the pathogenesis of various diseases. HNE is formed as a racemic mixture of (R)- and (S)- enantiomers. These enantiomers differ in their biological properties. The aim of this study was to investigate separately the in vivo metabolism of the two HNE enantiomers in male rats after intravenous administration of the corresponding radiolabeled compounds and to compare the results with those obtained with the racemic mixture. Although the difference in the excretion rates was not statistically significant, the HPLC profiles of urinary metabolites showed qualitative and quantitative differences between the two enantiomers. The level of 3-mercapturic acid-1,4-dihydroxynonane, which is considered as the major urinary metabolite of HNE, was significantly lower in the case of (S)-HNE injected rats. In vitro studies using rat liver cytosolic incubations and HNE-glutathione conjugate as substrate were performed to clarify the intermediate pathways involved in their metabolism. Large differences were obtained in the reduction and retro-Michael conversion steps of the metabolism between the conjugates originating from the two enantiomers.