Biomedical subjects
J M Guerrero
Publications and source records attributed to J M Guerrero.
Melatonin is responsible for the nocturnal increase observed in serum and thymus of thymosin alpha1 and thymulin concentrations: observations in rats and humans.
This paper shows that melatonin regulates both thymosin alpha1 and thymulin production as well as the expression of the prothymosin alpha gene. The results revealed the following facts: (a) The concentrations of thymosin alpha1 in both serum and thymus of rat showed a nyctohemeral profile with peak values late at night and basal values during the day. The concentrations of thymulin in rat serum also showed a 24-h rhythm with an increase in their values at night. This rhythmical character for thymosin alpha1, and thymulin was also found in the human serum. (b) Rats injected with melatonin during the day exhibited a significant increase in the concentrations of both peptides. Moreover, continuous light exposure on the animals at daytime and pinealectomy cause a decrease in thymosin a1 and thymulin concentrations with regards to those found in control rats. (c) Melatonin regulates the expression of the prothymosin alpha gene, analyzed by Northern blot. These results suggest that melatonin may be involved in the regulation of immune functions by increasing the thymic peptides production.
Functional and molecular characterization of VIP receptor--effector system in rat developing immunocompetent cells: G protein involvement.
Changes in the functional characteristics for vasoactive intestinal peptide (VIP) receptor-effector system were evaluated in rat developing immunocompetent cells (from 1-week-old animals up to 12-week-old animals). These characteristics include [125I]VIP binding studies, cell cyclic AMP (cAMP) generation, analysis of [125I]VIP-receptor complexes by cross-linking experiments, as well as developed-associated G proteins assayed by cholera and pertussis toxin-catalyzed ADP-ribosylation and Western blot. The Scatchard analysis of binding data was consistent with the existence of two classes of VIP binding sites with K(d) values unaltered and B(max) increased during postnatal development. The efficiency of VIP stimulation of cAMP generation increased from 1-week-old rats to adult conditions. The VIP-receptor complex apparent molecular mass (52-55 kDa) remains unaltered, but it was significantly lower in 2-week-old than in 8-week-old rats. ADP-ribosylated material by cholera toxin (CTx) was higher from 8-week-old than from 2-week-old animals, while ADP-ribosylation by pertussis toxin (PTx) was quantitatively higher in 8-week-old rats. Results were confirmed when immunoblots for different G protein subunits were performed.
Immunobiology of vasoactive intestinal peptide (VIP)
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Ototoxicity caused by aminoglycosides is ameliorated by melatonin without interfering with the antibiotic capacity of the drugs.
The production of free radicals seems to be involved in the mechanisms of ototoxicity. Aminoglycosides produce ototoxicity, which can be determined through distortion product otoacoustic emissions (OAEs) that measure the activity of the outer hair cells of the organ of Corti. An ototoxic chart was obtained in rats using gentamicin or tobramycin. Together with this treatment, the animals ingested melatonin in the drinking water, or melatonin was injected subcutaneously or intramuscularly. The distortion product OAEs were determined over a prolonged period of time for each of the groups. The effect of melatonin on the antibiotic capacity of the aminoglycosides used was also studied. Antibiograms inoculated with Escherichia coli or Pseudomonas aeruginosa and treated with gentamicin or tobramycin in the presence or absence of melatonin at quantities from pharmacological to physiological doses were performed. The ototoxicity produced by gentamicin and tobramycin was maximal from days 3 to 5 post-treatment, returning to normal values in 2 wk. When melatonin was present, the recovery was at day 5 post-treatment, independently of the means of administration of the pineal product. The antibiograms showed that melatonin had no effect on the antibiotic capacity. It is concluded that the ototoxicity caused by gentamicin and tobramycin is ameliorated by melatonin and that the pineal hormone does not interfere with the antibiotic capacity of these antibiotics.
Correlation between nuclear melatonin receptor expression and enhanced cytokine production in human lymphocytic and monocytic cell lines.
The report shows that melatonin enhances IL-2 and IL-6 production by two human lymphocytic (Jurkat) and monocytic (U937) cell lines via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZRalpha, RORalpha1 and RORalpha2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mtl receptor. In the absence of the membrane receptor, melatonin still activates IL-2 production. U937 cells express only the mtl receptor. Although melatonin binds to both U937 nuclei and membranes, CGP 52608, a ligand of the nuclear receptor for melatonin, does not inhibit melatonin binding to U937 nuclei, suggesting that a protein other than the RZR/RORalpha receptor was involved in the process. In U937 cells, melatonin did not modify basal production of IL-6 or when activated by PMA plus LPS (lipopolysaccharide), a treatment that downregulates the expression of the mtl receptor. However, in U937 cells activated with IFN-gamma, which induces the expression of the RORgamma1 and RORalpha2 nuclear receptors and represses the expression of the mt1 receptor, melatonin can activate IL-6 production. These results show that the expression of nuclear melatonin receptor is sufficient for melatonin to activate cytokine production in human lymphocytic and monocytic cell lines.
Ototoxicity caused by cisplatin is ameliorated by melatonin and other antioxidants.
The mechanism of the ototoxicity caused by cisplatin is based in the generation of reactive oxygen species, which interferes with the antioxidant protection of the organ of Corti. Conversely, the protection of the cochlea with antioxidants ameliorates the ototoxicity by cisplatin. The ototoxicity produced by cisplatin can be reversible or persistent, depending on the age of the patient, cumulative doses, number of chemotherapy cycles, history of noise exposure, and deteriorating renal function. We have obtained in rats an ototoxic chart utilizing cisplatin (10 mg/kg body weight injected intraperitoneally, once only). Together with this treatment, the animals were treated with melatonin in the drinking water (10 mg/L) or injected subcutaneously (250 microg), and with an antioxidant mixture, injected subcutaneously, composed of 0.25 mg alpha-tocopherol acid succinate, 3 mg ascorbic acid, 1 mg glutathione, and 60 mg N-acetylcysteine. The distortion product otoacoustic emissions were determined for a prolonged period of time for each animal. The ototoxicity produced by cisplatin was maximal from days 7 to 10 post-treatment, returning to normal values in a month. When melatonin and the antioxidant mixture were present, the recovery was between days 10 and 15 post-treatment, independent of the means of administration of the pineal product. We conclude that the ototoxicity caused by cisplatin is ameliorated by melatonin and other antioxidants.
Neuroprotection by melatonin from glutamate-induced excitotoxicity during development of the cerebellum in the chick embryo.
This work investigated the ability of melatonin to prevent cell damage in the cerebellar cortex of chick embryo caused by glutamate administration. Cell injury was evaluated estimating, at ultrastructural level, the phenomenon of cell death and the synaptogenesis of the Purkinje cells and the cerebellar glomerular synaptic complex. Administration of glutamate during cerebellar development of the chick provokes excitotoxic neuronal degeneration characterized by a phenomenon of neuronal cell death that exhibits essentially the features of a death pattern described as necrosis and the deletion of synaptogenic processes. Our results show that melatonin has a neuroprotective effect against glutamate-induced excitotoxicity. This effect is morphologically revealed by the lack of neural cell death in the embryos treated with melatonin prior to glutamate injection and also by the degree of a synaptogenesis similar to that exhibited by the control group. Likewise, we corroborate the absence of teratological effects of melatonin on chick cerebellar development. Although the possible mechanisms involved in the neuroprotective effect of melatonin are discussed, i.e., direct antioxidant effects, up-regulating endogenous antioxidant defenses, and inhibiting nitric oxide formation activated by glutamate, further studies are required to establish the actual mechanism involved in the neuroprotective effect of melatonin.
Serum cholesterol and lipid peroxidation are decreased by melatonin in diet-induced hypercholesterolemic rats.
The purpose of this study was to investigate the effect of melatonin, at pharmacological doses, on serum lipids of rats fed with a hypercholesterolemic diet. Therefore, different groups of animals were fed with either the regular Sanders Chow diet or a diet enriched in cholesterol. Moreover, animals were treated with or without melatonin in the drinking water for 3 months. We show that melatonin treatment did not affect the levels of cholesterol or triglycerides in rats fed with a regular diet. However, the increase in total cholesterol and low-density lipoprotein (LDL)-cholesterol induced by a cholesterol-enriched diet was reduced significantly by melatonin administration. On the other hand, melatonin administration prevented the decrease in high-density lipoprotein (HDL)-cholesterol induced by the same diet. No differences in the levels of very low-density lipoprotein (VLDL)-cholesterol and triglycerides were found. We also found that melatonin administration slightly decreased serum uric, bilirubin and increased serum glucose levels. Other biochemical parameters, including total proteins, creatinine, urea, phosphorus, calcium, glutamic oxalacetic transaminase (GOT), glutamic pyruvic transaminase (GPT), gamma-glutamyltranspeptidase (gamma-GT), acetyl cholinesterase (AcCho), and alkaline phosphatase (ALP) were not modified by melatonin treatment. Finally, lipid peroxidation (LPO) was studied in membranes of liver, brain, spleen, and heart as an index of membrane oxidative damage. Results show that hypercholesterolemic diet did not modify the LPO status in any of the tissues studied. However, chronic melatonin administration significantly decreased LPO. Results confirm that melatonin participates in the regulation of cholesterol metabolism and in the prevention of oxidative damage to membranes.
Identification of G-protein coupled receptor subunits in normal human dental pulp.
To respond appropriately to their environment, dental pulp cells must integrate informational input from multiple ligands, such as neuropeptides, growth factors, and vasoactive amines. These ligands act through multiple intracellular signaling pathways. G-protein coupled receptor subunits play a major role in this process, providing a mechanism for coordinated regulation of both messengers and effectors. Increasing number of neuropeptides have been found in pulpal tissue. However, there is no data about molecular identification of G-protein subunits in human dental pulp. To identify the postreceptor mechanism involved in dental pulp cell signal transduction, we performed a Western blot analysis of different G-protein subunits. Biopsy specimens of human dental pulp were prepared and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by analysis with appropriate antibodies. We detected G alpha q/alpha 11, short and long forms of G alpha s, beta common, Gio-3, and Gil-2 antigens with a molecular weight approximately 42 kDa, 42 and 45 kDa, 36 kDa, 40 kDa, and 40 kDa, respectively. These results indicate that human pulp cells possess the cellular machinery to respond to sensory neuropeptides when they are released from the peptidergic nerve fibers. On this basis, the relationships of postdevelopmental, age-dependent, and pathophysiological disorders of G-proteins subunits in dental pulp could be studied.
Involvement of nuclear receptors in the enhanced IL-2 production by melatonin in Jurkat cells.
This report shows that melatonin enhances IL-2 production by Jurkat cells via a nuclear receptor-mediated mechanism. Jurkat cells express nuclear (RZR alpha, ROR alpha 1, and ROR alpha 2) and membrane (mt1) melatonin receptors, and melatonin binds to Jurkat nuclei and membranes with the same affinity described for human peripheral blood mononuclear cells (PBMCs). Melatonin enhances IL-2 production by Jurkat cells activated by either phytohemagglutinin (PHA) or phorbol myristate acetate (PMA). PHA activation of Jurkat cells does not change the profile of melatonin receptor expression; on the contrary, PMA activation negatively regulates the mt1 receptor. In the absence of the membrane receptor, melatonin still activates the IL-2 production. These results show that the expression of the nuclear melatonin receptor is sufficient for melatonin to activate IL-2 production by Jurkat cells.
Postnatal development of vasoactive intestinal peptide receptor-effector system in rat immunocompetent cells.
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Characterization of membrane melatonin receptor in mouse peritoneal macrophages: inhibition of adenylyl cyclase by a pertussis toxin-sensitive G protein.
Melatonin binding sites were characterized in mouse peritoneal macrophages. Binding of 2-[125I]melatonin by macrophages fulfills all criteria for binding to a receptor site. Thus, binding was dependent on time, temperature and cell concentration, stable, reversible, saturable and specific. Stoichiometric studies showed a high-affinity binding site with a Kd of 0.58-0.71 nM. These data are in close agreement with data obtained from kinetic studies (Kd = 0.29 nM). The affinity of these binding sites suggests that they may recognize the physiological concentrations of melatonin in serum. Moreover, binding experiments using macrophage crude membranes showed that melatonin bound specifically to the membranes. Additionally, in competition studies we observed a low-affinity binding site (Kd = 2.02 microM). Melatonin inhibited significantly forskolin-stimulated cyclic AMP accumulation in a dose-dependent manner. This effect was blocked by luzindole, an antagonist of the melatonin membrane receptor. Pretreatment of macrophages with pertussis toxin blocked the inhibitory effect of melatonin. Pertussis toxin ADP-rybosilation and Western blot experiments demonstrated both alpha(i1/2) and alpha(i3/o) G protein subunits expression in mouse peritoneal macrophages membranes. Our results demonstrate the existence of melatonin receptors in mouse peritoneal macrophages, and a pertussis toxin-sensitive melatonin signal transduction pathway that involves the inhibition of adenylyl cyclase.
Melatonin activates Th1 lymphocytes by increasing IL-12 production.
Melatonin could act on immune system by regulating cytokine production of immunocompetent cells. The hormone enhances IL-2, IFN-gamma and IL-6 production by cultured human mononuclear cells. As enhancement of IL-6 production is related to monocyte activation by melatonin, the hormone acts on human lymphoid cells causing a Th1-type response. This paper shows that melatonin seems to promote a Th1-response by increasing IL-12 production. The hormone enhances IL-12 production by cultured monocytes under suboptimal stimulation in a dose-dependent way. The effect of the hormone increases when PBMCs are incubated with melatonin before monocyte isolation. Enhanced IL-12 production by melatonin can also be shown in cultured human mononuclear cells.
In vitro effect of the resin component bisphenol A on substrate adherence capacity of macrophages.
This study was design to investigate the "in vitro" effect of bisphenol A (BPA), a component of resin used in dentistry, on viability, and substrate adherence capacity of macrophages. Peritoneal macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. Viability was determined by trypan blue exclusion. As a test of macrophage adhesion, the adherence capacity of macrophages to a plastic surface was determined and the adherence index was calculated. Assays were conducted in Eppendorf tubes for 60 min of incubation at 37 degrees C in a humidified atmosphere of 5% CO2 in air. BPA did not alter significantly macrophage viability at concentrations as high as 10(-5) M, but BPA decreased in a dose-dependent manner the adherence index of rat peritoneal macrophages. Control peritoneal macrophages showed an adherence index = 81.5 +/- 7.9%. In the presence of 10(-8) M BPA, the Al of macrophages decreased to 41.4 +/- 12.2% (p < 0.05). Higher BPA concentrations (10(-7) to 10(-5) M) also caused a significant inhibition of the adherence index. Half-maximal inhibition (IC50) was obtained at 4.92 +/- 0.39 x 10(-6) M BPA. The in vitro study shows that the resin component BPA can alter macrophage adhesion. Taking into account that adhesion is the first step in the phagocytic process of macrophages and in antigen presentation, BPA could inhibit macrophage function and modulate immune and inflammatory responses in dental pulp and periapical tissues.
Comparative effects of two endodontic irrigants, chlorhexidine digluconate and sodium hypochlorite, on macrophage adhesion to plastic surfaces.
This study was designed to compare the "in vitro" effect of chlorhexidine digluconate, proposed as a new irrigant solution, with sodium hypochlorite, the currently irrigant solution used to disinfect the root canal system before obturation of the canal, on substrate adherence capacity of macrophages. Inflammatory macrophages were obtained from Wistar rats and resuspended in RPMI-1640 medium. The adherence capacity of macrophages to a plastic surface was determined. Assays were conducted in Eppendorf tubes for 15 min of incubation at 37 degrees C in an humidified atmosphere of 5% CO2 in air. The adherence index was calculated. Chlorhexidine digluconate inhibited substrate adherence capacity of macrophages in all conditions tested. Half-maximal inhibition (IC50) was observed at 1:5.6 dilution. Chlorhexidine digluconate was less potent than 5.25% sodium hypochlorite (IC50 = 1:24.1 dilution) in inhibiting substrate adherence capacity of macrophages. Taking into account that substrate adherence is the first step in the phagocytic process of macrophages and in antigen presentation, chlorhexidine digluconate could inhibit macrophage function and modulate inflammatory reactions at the level of inflamed periapical tissues.
The pineal secretory product melatonin reduces hydrogen peroxide-induced DNA damage in U-937 cells.
Melatonin, the chief secretory product of the pineal gland, is a potent and efficient endogenous radical scavenger. Thus, melatonin was shown to protect different biomolecules, such as DNA, membrane lipids, and cytosolic proteins, from oxidative damage induced by oxygen-derived free radicals. In order to study the protective role of melatonin in hydrogen peroxide (H2O2)-induced DNA damage, U-937 cells were treated with different concentrations of H2O2, either in the presence or absence of melatonin, and DNA damage was assessed using the cytokinesis-block micronucleus technique. Melatonin diminished H2O2-induced micronuclei production both in short and long treatments. Additionally, melatonin concentrations higher than 1 microM were capable of protecting cells from spontaneous micronuclei production. These data suggest that melatonin, an endogenous antioxidant and nontoxic compound, may have an important role in protecting cells from genetic damage due to free radicals, supporting the idea of this hormone as a possible therapeutic agent in preventing aging and age-related diseases.
Physiological levels of melatonin contribute to the antioxidant capacity of human serum.
This work evaluates whether physiological concentrations of the pineal secretory product melatonin contribute to the total antioxidant status (TAS) of human serum. Day and nighttime serum samples were collected from healthy volunteers ranging from 2 to 89 years of age and used to measure melatonin and TAS. Results showed that both melatonin and TAS in human serum exhibited 24 hr variations with nocturnal peak values at 01:00 hr. Moreover, exposure of volunteers to light at night resulted in clear decreases of both TAS and melatonin. Furthermore, when melatonin was removed from sera collected at night, the TAS value of the sample was reduced to basal daytime values. In aging studies, it was found that nocturnal serum values of TAS and melatonin exhibited maximal values during the first four decades; thereafter, these values decreased as age advanced. In 60-year-old individuals, day/night differences in serum melatonin and TAS levels were clearly diminished, by more than 80%, with these differences being completely abolished in older individuals. Our results suggest that melatonin contributes to the total antioxidative capability of human serum. This antioxidant contribution of melatonin is reduced as age advances correlating with the age-related reduction of melatonin.