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J Peinado

Publications and source records attributed to J Peinado.

At least 19 recordsLinked to original sources
PubMed2005

Ecotoxicological effects of metal pollution in two mollusc species from the Spanish South Atlantic littoral.

Metal accumulation and some of their biochemical effects have been studied in oysters (Crassostrea angulata) and mussels (Mytilus galloprovincialis) of the South Atlantic Spanish littoral. Especial attention has been paid to antioxidant defences and oxidative damage to biomolecules. Deep differences in the response of oysters and mussels to metal pollution were found. Oysters, with the higher metal loads of both species, showed increased antioxidant defences, and less extensive oxidative damage. In contrast, mussels, which accumulated much lower metal concentrations, showed clear increases in oxidized biomolecules, in agreement with their low increases in the antioxidant defence mechanisms. Our results suggest that mussels are more sensitive and less well adapted to metal pollution, probably explaining their absence in the most contaminated studied site, Mazagón. We conclude that oysters can be used as more sensitive bioindicator of pollution in the South Spanish littoral, and as a suitable model to study the adaptation to metal pollution.

Animals↗
PubMed2003

Effect of food deprivation on oxidative stress biomarkers in fish (Sparus aurata).

Oxidative stress in fish (Sparus aurata) as a consequence of food restriction and fasting, has been studied. Four groups of fish were maintained for 46 days under different conditions of food supplementation: a control group with no food restriction (ratio of food/fish of 2% w/w), two groups of animals with restricted food supplement (1 and 0.5%) and a fasting group (no meal addition). Finally, all the fish were provided with food at the same ratio as the control group for the last 7 days. Sampling and weighing of fish were carried out every week and their livers were used for the analysis of known biomarkers of oxidative stress. Malondialdehyde and oxidized glutathione levels increased at the third week in fish with partial or total food deprivation, but these levels returned to normal values when the fish readapted to the control conditions. Antioxidant enzymes were also analyzed and significant increases in superoxide dismutase (SOD), glutathione reductase and glutathione peroxidase activities were found in parallel with food restriction; however catalase activity decreased in fasting fish. New SOD isoforms were detected by isoelectrofocusing in fish under food restriction at the second week, which disappeared when starved fish returned to the control conditions. These new SOD isoforms were detected before the appearance of other usual oxidative stress biomarkers.

Animals↗
PubMed2003

Reduction of extracellular dopamine and metabolite concentrations in rat striatum by low doses of acute cyamemazine.

The low incidence of extrapyramidal effects with atypical neuroleptics has been ascribed to their 5-HT(2A)- and 5-HT(2C)-serotonin receptor antagonistic properties. On the other hand, the acute increase in striatal dopamine release by submaximal dopamine D(2) autoreceptor blockade can be respectively reduced and increased by 5-HT(2A)- and 5-HT(2C)-antagonists. Cyamemazine is a neuroleptic D(2)- and 5-HT(2A)-receptor antagonist, with small antagonistic activity at 5-HT(2C) receptors and low incidence of extrapyramidal side effects. Therefore, submaximal cyamemazine was tested in rats for its acute action on the extracellular concentrations of dopamine and dopamine metabolites (DOPAC: 3,4,dihydroxyphenylacetic acid and HVA: 4-hydroxy-3-methoxy-phenyl-acetic acid) in the corpus striatum. The serotonin metabolite 5-HIAA (5-hydroxy-indole-acetic acid) was measured in parallel. Rats prepared for microdialysis (striatum) were intraperitoneally given cyamemazine 1 mg/kg, 5 mg/kg or vehicle ( n=4 in each group). Dopamine, DOPAC, HVA and 5-HIAA concentrations in perfusates under basal conditions and after stimulation by high K(+) were measured by HPLC coupled to electrochemical detection. Cyamemazine 1 mg/kg significantly reduced extracellular concentrations of basal dopamine (-77%), DOPAC (-54%), HVA (-54%) and 5-HIAA (-65%). No such effects were seen with the dose of cyamemazine 5 mg/kg or for K(+)-evoked dopamine release. In conclusion, submaximal cyamemazine can acutely reduce basal dopamine release and metabolism in the rat striatum. Such unusual action can be explained by the original pharmacological profile of cyamemazine (potent D(2)- and 5-HT(2A)-antagonist, with small antagonistic activity at 5-HT(2C) receptors). Further experiments are required to explain the low incidence of extrapyramidal side actions with cyamemazine.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1998

Incubation of superoxide dismutase with malondialdehyde and 4-hydroxy-2-nonenal forms new active isoforms and adducts. An evaluation of xenobiotics in fish.

The effects in fish (Sparus aurata) of dieldrin, previously reported to be an inducer of peroxisomal enzymes (Pedrajas et al., Comp. Biochem. Physiol. 115C (1996) 125-131), were compared with those of clofibrate. Although dieldrin provoked the more severe peroxisomal changes, both compounds induced oxidative stress as detected by the increased levels of microsomal thiobarbituric acid reactive substances; however the malondialdehyde (MDA) content, determined after HPLC separation of the MDA-TBA complex, was not significantly altered. These results suggest that, besides MDA, other aldehydes were formed in xenobiotic-injected fish, leading us to assess the oxidative effects of such xenobiotics by following changes in superoxide dismutase (SOD) pattern. New active SOD isoforms were detected by isoelectrofocusing in the light mitochondrial (LMF) and cytosolic (CF) fractions. Most of the new SOD bands could be reproduced in vitro by incubation of fish liver cell-free extracts with MDA. To clarify the effects of aldehydes, Cu,Zn- and Mn-SOD isoforms were purified and amino acid analysis was carried out. The new bands found in LMF and CF fractions were reproduced in vitro after incubation of pure SODs with MDA and 4-hydroxy-2-nonenal (HNE), the new SOD bands formed being coincident with the loss of Lys or His residues. Lysine residues were preferentially derivatized after treatment of Cu,Zn-SOD with MDA, but in Mn-SOD the lysine residues were modified only after treatment with MDA, while the histidine residues were modified only by HNE. No change of SOD activity was detected after MDA or HNE exposure, although at the higher aldehyde concentrations used protein aggregates were formed. Therefore, the appearance of new active SOD bands, after isoelectrofocusing separation, can be proposed as a biomarker of oxidative stress.

Aldehydes↗
PubMed1998

The carboxy-terminal region of human lipoprotein lipase is necessary for its exit from the endoplasmic reticulum.

Certain missense substitutions on the human lipase (hLPL) gene produce mutated proteins that are retained in different compartments along the secretory pathway. The purpose of the present study was to elucidate whether the C-terminal domain of the hLPL molecule could be important for secretion. We constructed by site-directed mutagenesis three carboxy-terminal mutated (F388-->Stop, K428-->Stop and K441-->Stop) hLPL cDNAs that were expressed in COS1 cells. Immunoblotting of cell extracts showed that all three constructs led to similar levels of protein. Both wild type (WT) hLPL and the truncated K441-->Stop hLPL were secreted to the extracellular medium, and presented a similar intracellular distribution pattern as shown by immunofluorescence. Neither F388-->Stop nor K428-->Stop hLPL protein was detected in cell medium. Immunofluorescence experiments showed that both truncated hLPL were retained within an intracellular compartment, which became larger. Double immunofluorescence analysis using antibodies against LPL and antiprotein disulfide isomerase as a marker showed that the truncated K428-->Stop hLPL was retained within the rough endoplasmic reticulum. This truncated protein was not found in other compartments in the secretory pathway, such as Golgi complex and lysosomes, indicating that it did not exit the endoplasmic reticulum. Further analysis of the C-terminal region of the LPL molecular model showed both that F388-->Stop and K428-->Stop hLPL truncated proteins are highly hydrophobic. As retention of secretory proteins in the rough endoplasmic reticulum is a quality control mechanism of the secretory pathway, we conclude that the C-terminal domain of hLPL is critical for correct intracellular processing of the newly synthesized protein.

DNA Mutational Analysis↗
PubMed1996

Dieldrin induces peroxisomal enzymes in fish (Sparus aurata) liver.

We have previously described the increase of microsomal lipid peroxidation and the appearance of new oxidized forms of Cu,Zn-superoxide dismutase in the liver of gilthead seabrams (Sparus aurata) injected with model xenobiotics, due to the increased production of reactive oxygen species (ROS) (Pedrajas et al., Chem. Biol. Interact., 1995). The effects of dieldrin and copper(II) on subcellular organelles directly related with ROS production are now studied. Immature fish were injected with dieldrin and copper, 0.15 and 1.0 mg/ kg, respectively. After 2 and 7 days, the livers were homogenized and the catalase and superoxide dismutase activities were determined in subcellular fractions isolated by differential centrifugation: nucleus, mitochondria, light mitochondrial fraction (LMF), microsomes and cytosolic fraction. Peroxisomes were isolated from LMF by discontinuous gradient centrifugation using Nycodenz. Changes in catalase and superoxide dismutase depended on the xenobiotic and affected to different subcellular fractions. Thus, the effects of copper(II) were mainly in nucleus and cytosol, whereas dieldrin induced catalase and superoxide dismutase (up to 2.8-fold) preferentially in nucleus and LMF fractions. Dieldrin-injected fish showed also highly increased activity of palmitoyl-CoA-oxidase (9.3-fold) and a nearly 2-fold increase in the protein concentration of the peroxisomal fraction. The results described above suggest that the oxidative stress previously detected for dieldrin in fish liver affects also to the peroxisomal enzymes.

Animals↗
PubMed1996

Oleoyl-estrone induces the loss of body fat in rats.

OBJECTIVE: Four experiments were devised to test the possible role of estrone fatty esters as adipose tissue signals carried by the blood within lipoproteins. DESIGN: Oleoyl-estrone was synthesized and incorporated in liposomes; it was administered i.v. (to mimic lipoprotein delivery) for 14-day periods using implantable osmotic minipumps. The study included the finding of oleoyl-estrone in blood lipoproteins, the correlations of the effects of body weight to the dose and the uptake of labelled oleoyl-estrone by tissues, its internalization and disposal. SUBJECTS: Normal-weight Wistar female rates were used. Pooled human blood was used as source of HDL3. MEASUREMENTS: Oleoyl-estrone was identified in rat white adipose tissue and in human blood HDL3 lipoprotein fraction. Changes in body weight, food intake, oxygen consumption, respiratory quotient and nitrogen balance were measured in chronically injected rats. The uptake and hydrolysis of oleoyl-estrone by tissues was also determined following its acute administration. RESULTS: Oleoyl-estrone induced a dose-dependent loss of weight, with decreased food intake. In 14 days, and compared with controls at the end of this period, a dose of 0.78 mumol/day induced the loss of 16.4 +/- 5.5% of body weight; the difference was maximal for doses of 15 mumol/day or higher: 24.7 +/- 3.1%. Under oleoyl-estrone treatment, body protein was preserved (positive nitrogen balances) and fat stores were wasted: lowered respiratory quotient, and deficit in energy balance; a dose of 0.78 mumol/day induced the loss of 9.6 +/- 2.2 g of total body lipids in 14 days. Most of oleoyl-estrone taken up by tissues was hydrolysed; however, in part it reached intact the cell nucleus of incubated adipocytes. Oleoyl-estrone effects were different from those of free estrone. CONCLUSION: A lipophilic pathway for oleoyl-estrone transport by lipoproteins is postulated, allowing chemical communication between tissues. Oleoyl-estrone may be directly involved in the control of body weight.

Adipocytes↗
PubMed1995

Oxidative stress in fish exposed to model xenobiotics. Oxidatively modified forms of Cu,Zn-superoxide dismutase as potential biomarkers.

Fish (Sparus aurata) were intraperitoneally injected with model xenobiotics and several biomarkers of oxidative stress were analysed after 2 and 7 days exposure. The levels of soluble thiobarbituric acid reactive substances (TBARS) increased markedly in animals treated with polar xenobiotics, CuCl2 or paraquat; exposure to the apolar xenobiotics, dieldrin or malathion, enhanced significantly the microsomal TBARS while decreasing the microsomal glutathione transferase activity. The specific superoxide dismutase (SOD) activity increased in Cu(II)-injected animals but diminished in fish exposed to paraquat. After isoelectrofocusing separation and activity staining cell-free extracts of fish exposed to Cu(II), dieldrin or malathion displayed two new Cu,Zn-SOD isoforms of intermediate pI. An additional Mn-SOD was observed in dieldrin-injected fish, but only a faint new acidic isoform was observed in paraquat-injected animals. The new SOD bands were reproduced in vitro by incubation of cell-free extracts with systems generating superoxide anion or hydrogen peroxide and with a tert-butyl hydroperoxide/ADP-Fe system. Metallothionein induction was observed in Cu(II) or paraquat-exposed fish, but not in animals injected with apolar xenobiotics. So, the new SOD bands are possibly oxidized forms of this enzyme and can be considered as useful early biomarkers of oxidative stress due to transition metals or organic xenobiotics.

Animals↗
PubMed1995

The mutant Asn291-->Ser human lipoprotein lipase is associated with reduced catalytic activity and does not influence binding to heparin.

Lipoprotein lipase (LPL) plays a central role in triglyceride metabolism, regulating the catabolism of triglyceride-rich lipoprotein particles. LPL performs its hydrolytic action attached to heparan sulfate proteoglycans at the luminal surface of capillary endothelial cells. We have assessed the effect of the Asn291-->Ser (N291S) substitution found in LPL gene from a human hyperlipemic patient. Our results showed that both the wild-type (WT) and N291S hLPL expressed in COS1 cells were secreted to the extracellular medium, and presented similar intracellular distribution patterns. Furthermore, heparin-Sepharose affinity chromatography assays revealed normal heparin affinity of the N291S hLPL. In addition, both the mutant and the WT protein bound to the surface of human fibroblasts and untransfected COS1 cells. Interestingly, diminished LPL specific activity was observed in the extracellular medium from mutant expressing cells. Therefore the lack of normal LPL activity in patients harbouring such a mutation could be the cause of their hyperlipemic disorder.

Base Sequence↗
PubMed1993

Biochemical and genetic indices of marine pollution in Spanish littoral.

Increased activities of several detoxifying and antioxidant enzymes were detected in mollusc and fish from Spanish littoral areas with high metal contents. Ethanolic extracts from molluscs contained direct-acting and polar genotoxins of oxidative type, which were detected by strain TA102 of S. typhimurium and catalase-deficient strains of E. coli. Animals from contaminated sites contained less genotoxins than those from control areas. Polluted fishes displayed highly induced cytochrome P-450 activity and increased promutagen activation capabilities. In addition, specific forms of glutathione transferase and superoxide dismutase were induced, particularly highly acidic forms.

Animals↗
PubMed1993

Purification of Cu, Zn-superoxide dismutase isoenzymes from fish liver: appearance of new isoforms as a consequence of pollution.

Liver cell-free extracts of fish (Mugil sp.) from polluted environments show new Cu,Zn-SOD isoenzymes when analyzed by polyacrylamide gel electrophoresis or isoelectrofocusing followed by in situ staining for SOD activity. The most active isoenzymes, with pI 6.1 and 5.1, were present both in control and problem samples while the isoenzymes of intermediate pI value showed significant differences. Fish from control areas showed three intermediate isoenzymes with pI 5.7, 5.5 and 5.4 (the last one quite faint) while polluted animals showed three bands of pI 5.9, 5.45 and 5.35, this last very intense. To further characterize their utility as biomarkers, Cu,Zn-SOD isoenzymes from polluted fish livers were purified to homogeneity. Five superoxide dismutase peaks were purified, named thereafter I (pI 6.1) to V (pI 5.1) respectively. Isoenzymes I and V displayed the highest specific activity. Upon incubation with moderate H2O2 concentrations, pure isoenzyme I yielded more acidic bands with pI 5.5, 5.45 and 5.35, this last being predominant. The pure isoenzyme V generated only a new band of pI 5.0. Concomitant with oxidation, the activity of peaks I and V was lost in a H2O2 concentration-dependent manner. The pattern of the new acidic bands generated upon the oxidixing treatment of isoenzyme I closely resembles that observed in crude extracts from polluted animals.

Animals↗
PubMed1992

Glutathione reductase from Saccharomyces cerevisiae undergoes redox interconversion in situ and in vivo.

Redox interconversion of glutathione reductase was studied in situ with S. cerevisiae. The enzyme was more sensitive to redox inactivation in 24 hour-starved cells than in freshly-grown ones. While 5 microM NADPH or 100 microM NADH caused 50% inactivation in normal cells in 30 min, 0.75 microM NADPH or 50 microM NADH promoted a similar effect in starved cells. GSSG reactivated the enzyme previously inactivated by NADPH, ascertaining that the enzyme was subjected to redox interconversion. Low EDTA concentrations fully protected the enzyme from NADPH inactivation, thus confirming the participation of metals in such a process. Extensive inactivation was obtained in permeabilized cells incubated with glucose-6-phosphate or 6-phosphogluconate, in agreement with the very high specific activities of the corresponding dehydrogenases. Some inactivation was also observed with malate, L-lactate, gluconate or isocitrate in the presence of low NADP+ concentrations. The inactivation of yeast glutathione reductase has also been studied in vivo. The activity decreased to 75% after 2 hours of growth with glucono-delta-lactone as carbon source, while NADPH rose to 144% and NADPH+ fell to 86% of their initial values. Greater changes were observed in the presence of 1.5 microM rotenone: enzymatic activity descended to 23% of the control value, while the NADH/NAD+ and NADPH/NADP+ ratios rose to 171% and 262% of their initial values, respectively. Such results indicate that the lowered redox potential of the pyridine nucleotide pool existing when glucono-delta-lactone is oxidized promotes in vivo inactivation of glutathione reductase.

Cell Membrane Permeability↗
PubMed1991

Metals are directly involved in the redox interconversion of Saccharomyces cerevisiae glutathione reductase.

Redox inactivation of glutathione reductase involves metal cations, since chelators protected against NADPH-inactivation, 3 microM EDTA or 10 microM DETAPAC yielding full protection. Ag+, Zn2+ and Cd2+ potentiated the redox inactivation promoted by NADPH alone, while Cr3+, Fe2+, Fe3+, Cu+, and Cu2+ protected the enzyme. The Zn2+ and Cd2+ effect was time-dependent, unlike conventional inhibition. Glutathione reductase interconversion did not require dioxygen, excluding participation of active oxygen species produced by NADPH and metal cations. One Zn2+ ion was required per enzyme subunit to yield full NADPH-inactivation, the enzyme being reactivated by EDTA. Redox inactivation of glutathione reductase could arise from the blocking of the dithiol formed at the active site of the reduced enzyme by metal cations, like Zn2+ or Cd2+. The glutathione reductase activity of yeast cell-free extracts was rapidly inactivated by low NADPH or moderate NADH concentrations; NADP+ also promoted rapid inactivation in fresh extracts, probably after reduction to NADPH. Full inactivation was obtained in cell-free extracts incubated with glucose-6-phosphate or 6-phosphogluconate; the inactivating efficiency of several oxidizable substrates was directly proportional to the specific activities of the corresponding dehydrogenases, confirming that redox inactivation derives from NADPH formed in vitro.

Anaerobiosis↗
PubMed1990

Effect of starvation on lipoprotein lipase activity in the liver of developing rats.

Liver lipoprotein lipase activity in neonatal (1- and 5-day-old) rats was 2-3-times than in the liver of adult rats. In mid-suckling (15-day-old) or weaned (30-day-old) animals, it was not significantly different from the low activity detected in adult rats. Starvation resulted in a 3-fold increase of lipoprotein lipase activity in the neonatal liver, but did not affect the activity in the liver of mid-suckling, weaned or adult rats. When isolated livers from both 1- and 5-day-old pups were perfused with heparin, a sharp peak of lipoprotein lipase activity appeared in the perfusate. In fed neonates, the peak area accounted for about 70% of the total (released + non-releasable) activity. In starved neonates, the proportion of heparin-releasable activity increased up to about 90%. These results indicate that neonatal rat liver lipoprotein lipase activity is markedly affected by changes in the nutritional status of the animal, and the effect is restricted to the vascular pool of the enzyme, as was reported in extrahepatic tissues from adult rats.

Animals↗
PubMed1989

Lipoprotein lipase activity in neonatal-rat liver cell types.

The lipoprotein lipase activity in the liver of neonatal (1 day old) rats was about 3 times that in the liver of adult rats. Perfusion of the neonatal liver with collagenase decreased the tissue-associated activity by 77%. When neonatal-rat liver cells were dispersed, hepatocyte-enriched (fraction I) and haemopoietic-cell-enriched (fraction II) populations were obtained. The lipoprotein lipase activity in fraction I was 7 times that in fraction II. On the basis of those activities and the proportion of both cell types in either fraction, it was estimated that hepatocytes contained most, if not all, the lipoprotein lipase activity detected in collagenase-perfused neonatal-rat livers. From those calculations it was also concluded that haemopoietic cells did not contain lipoprotein lipase activity. When the hepatocyte-enriched cell population was incubated at 25 degrees C for up to 3 h, a slow but progressive release of enzyme activity to the incubation medium was found. However, the total activity (cells + medium) did not significantly change through the incubation period. Cycloheximide produced a time-dependent decrease in the cell-associated activity. Heparin increased the amount of lipoprotein lipase activity released to the medium. Because the cell-associated activity was unchanged, heparin also produced a time-dependent increase in the total activity. In those cells incubated with heparin, cycloheximide did not affect the initial release of lipoprotein lipase activity to the medium, but blocked further release. The cell-associated activity was also decreased by the presence of cycloheximide in those cells. It is concluded that neonatal-rat hepatocytes synthesize active lipoprotein lipase.

Animals↗
PubMed1989

Hepatic endothelial lipase activity in neonatal rat liver.

Hepatic endothelial lipase (HEL) activity is as high in the neonatal (1-day old) rat liver as in adults. Most of the HEL activity is located at the capillaries since 75% of the total activity is released by heparin or collagenase perfusion. The residual activity (non-releasable) is located in hepatocytes and not in hemopoietic cells, which are the major cell type in neonatal liver. Per mg of protein, the HEL activity is 50% higher in neonatal than in adult hepatocytes. We suggest that neonatal hepatocytes have an increased capacity to synthesize and secrete HEL activity, so maintaining a high activity in the whole organ. It might contribute to the hepatic uptake of cholesterol from circulating lipoproteins, in a period in which endogenous cholesterol synthesis is known to be inhibited in the liver.

Animals↗
PubMed1989

Hepatic lipoate uptake.

Uptake of [35S]lipoate was studied in perfused rat liver and in isolated rat hepatocytes. During single-pass perfusion of [35S]lipoate about 30% of the radioactivity is retained in the liver. A substantial amount of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive material appears in the effluent perfusate, while hepatic efflux of GSH is unchanged. The hepatic uptake of lipoate, the release of thiols, and also the biliary excretion of 35S-labeled compounds are suppressed by octanoate. In isolated hepatocytes the uptake of lipoate follows saturation kinetics showing a Km value of 38 microM and a Vmax of 180 pmol/mg X 10 s. The uptake is temperature-dependent; from the Arrhenius plot an activation energy of 14.8 kcal/mol at 20 microM lipoate is calculated. At high concentrations of lipoate (above 75 microM) a nonsaturable uptake component becomes predominant. Lipoate uptake is selectively inhibited by medium-chain fatty acids. Only slight inhibition is seen in the presence of long-chain fatty acids, and there is no inhibition with acetate or lactate. Substantial inhibition is also observed with acetylsalicylic acid, but not with taurocholate, bromosulfophthalein or biotin. Lipoate uptake can be inhibited by high concentrations of phloretin (200 microM) and is rather insensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (200 microM). The results indicate that hepatic uptake of lipoate at physiological concentrations is largely carrier-mediated.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗