Biomedical subjects

L G Costa

Publications and source records attributed to L G Costa.

At least 73 records · Page 4Linked to original sources

PubMed1995

Ethanol inhibits muscarinic receptor-stimulated phosphoinositide metabolism and calcium mobilization in rat primary cortical cultures.

In recent years, it has been hypothesized that muscarinic receptor-stimulated phosphoinositide (PI) metabolism may represent a relevant target for the developmental neurotoxicity of ethanol. Age-, brain region-, and receptor-specific inhibitory effects of ethanol on this system have been found, both in vitro and after in vivo administration. As a direct consequence of this action, alterations of calcium homeostasis would be expected, through alterations of inositol trisphosphate formation, which mediates intracellular calcium mobilization. In the present study, the effects of ethanol (50-500 mM) on carbachol-stimulated PI metabolism and free intracellular calcium levels were investigated in rat primary cortical cultures, by measuring release of inositol phosphates and utilizing the two calcium probes fluo-3 and indo-1 on an ACAS (Adherent Cell Analysis and Sorting) Laser Cytometer. Ethanol exerted a concentration-dependent inhibition of carbachol-stimulated PI metabolism. In addition, ethanol's inhibitory effect paralleled the temporal development of the muscarinic receptor signal transduction system, with the strongest inhibition (25-50%) occurring when maximal stimulation by carbachol occurs (days 5-7). Ethanol also exerted a concentration-dependent decrease in free intracellular calcium levels following carbachol stimulation. Both initial calcium spike amplitude, seen in all responsive cells, as well as the total number of cells responding to carbachol, were decreased by ethanol. The inhibitory effects of ethanol seemed dependent upon preincubation time, in that a longer preincubation (30 min) with the lowest dose (50 mM), showed almost the same decrease in responding cell number and reduction in spike amplitude in responding cells, as a shorter incubation (10 min) with the highest ethanol dose (500 mM). The specificity of the response to carbachol was demonstrated by blocking the response with 10 microM atropine. Moreover, experiments with carbachol in calcium-free buffer with 1 mM EGTA indicated that the initial calcium spike was due to intracellular calcium mobilization from intracellular stores. Since calcium is believed to play important roles in cell proliferation and differentiation, these results support the hypothesis that this intracellular signal-transduction pathway may be a target for ethanol, contributing to its developmental neurotoxicity.

Action Potentials↗

PubMed1995

Interaction of ethanol and anoxia with muscarinic receptor--stimulated phosphoinositide metabolism during brain development.

The mechanism(s) by which ethanol induces alterations in brain development may involve direct actions (e.g. changes in specific biochemical pathways), or indirect effects, such as cerebral hypoxia resulting from ethanol - induced circulatory changes. Since both ethanol and hypoxia are known to affect the metabolism of phosphoinositides, which has been suggested as a possible target for ethanol's developmental neurotoxicity, in the present study we have investigated the in vitro effects of both severe hypoxia (anoxia) and ethanol (alone or in combination) on muscarinic receptor-stimulated phosphoinositide metabolism in cerebral cortex slices from neonatal rats. Anoxia markedly inhibited carbachol - stimulated phosphoinositide metabolism in adult rats (67%), but only slightly (10%) in neonatal animals. Reoxygenation reversed the effect of anoxia at both ages. On the other hand, ethanol's inhibitory effect was pronounced in neonatal rats only, and was additive to that of anoxia. The presence of ethanol did not affect the recovery of carbachol - stimulated phosphoinositide metabolism following anoxia and reoxygenation. These results indicate that ethanol and anoxia differently and independently affect muscarinic receptor - stimulated phosphoinositide metabolism and may mutually contribute to the CNS effects observed following developmental ethanol exposure.

Age Factors↗

PubMed1995

Identification of m3, m4 and m5 subtypes of muscarinic receptor mRNA in human blood mononuclear cells.

In this study we made use of the Reverse transcription-polymerase chain reaction to analyze the expression of mRNA for the five subtypes of muscarinic acetylcholine receptors in human blood mononuclear cells. mRNA for m3, m4 and m5 subtypes was detected, while mRNA for m1 and m2 muscarinic receptors was not found. Similar results were obtained for three different healthy human subjects studied. Interestingly, the m5 subtype was expressed at higher levels in blood mononuclear cells than in cerebral cortex. To our knowledge this is the first time that m5 muscarinic receptor mRNA has been found outside of the central nervous system.

Adult↗

PubMed1995

Mechanisms of neurotoxicity: applications to human biomonitoring.

Interactions of chemicals with cerebral neurotransmitters, receptors, and second messenger systems are often accompanied by similar changes involving components in non-neural tissues. On this basis, indirect strategies have been developed to investigate neural cell function parameters by methods using accessible cells such as platelets or peripheral blood lymphocytes. The validity of certain surrogate markers of biochemical events occurring in the nervous system has been documented by recent studies in both laboratory animals and humans. Although experience with neurotoxicants is still limited, advantages and limitations of methods using peripheral blood cells as indicators of chemically-induced nervous system changes have been documented by a number of studies in psychopharmacology and biological psychiatry. Applicability of this approach in conventional population studies of environmental chemicals remains to be demonstrated. However, recent data regarding the action of low doses of mercury and organophosphates on receptors and signal transduction pathways in peripheral lymphocytes suggest useful applications of certain surrogate markers in mechanistic studies of neurotoxicity in vivo and, possibly, in assessing early biochemical effects of neurotoxicants in humans.

Biomarkers↗

PubMed1995

Biochemical markers of neurotoxicity: research strategies and epidemiological applications.

A great deal of attention has been given in the past few years to the development and validation of biomarkers for non-cancer endpoints, to be used in human epidemiological studies. In this research, as they apply to the field of neurotoxicology, will be discussed. As biomarkers are often divided into indicators of exposure, effect and susceptibility, one example for each of these classes is presented. Measurements of hemoglobin adducts were developed as a way to monitor exposure to acrylamide (a peripheral neurotoxicant) in animals as well as humans, and have been successfully applied in a field study in occupationally exposed workers. Activity of monoamine oxidase B (MAO-B) in platelets was found to be inversely correlated with the levels of exposure to styrene, suggesting that this biochemical measurement may be a useful effect-related biomarker, though additional studies are needed to understand the mechanistic implications of these findings. Serum paraoxonase activity has been shown in animals to modulate the toxicity of organophosphorus insecticides. Studies in humans have lead to the identification of a genetic polymorphism for this enzyme and to define its underlying molecular basis. Epidemiological studies are needed to determine the relevance of this polymorphism in determining susceptibility to organophosphate toxicity.

Biomarkers↗

PubMed1995

Paraoxonase protects against chlorpyrifos toxicity in mice.

Paraoxonase can hydrolyze paraoxon (PO), chlorpyrifos-oxon (CPO) and other organophosphates. Previous studies have indicated that the levels of serum paraoxonase can influence the toxicity of PO and CPO. In the present study we have investigated whether exogenous paraoxonase administered to mice would offer protection toward the acute toxicity of a phosphorothioate, chlorpyrifos (CPS). Paraoxonase was purified from rabbit serum and injected i.v., or i.v. plus i.p., in mice. Inhibition of acetylcholinesterase (AChE) in brain, diaphragm, plasma and red blood cells was measured as an index of CPS (100 mg/kg) toxicity. Administration of paraoxonase 30 min before CPS increased plasma enzyme activity toward CPO by 35-fold, and protected against its toxicity; protection was still present at 24 h, when enzyme activity was still 20-fold over basal. When paraoxonase was given 30 min after CPS, a significant protection against CPS toxicity was still observed, while after 3 h the protective effect was decreased. To mimic conditions of severe acute poisoning, a higher dose of CPS (150 mg/kg) was also administered. Administration of paraoxonase 30 min after this exposure abolished cholinergic signs and significantly protected against AChE inhibition. These results indicate that exogenous paraoxonase offers significant protection against CPS toxicity when administered both before and after the organophosphate, suggesting that it may be considered as a potential additional treatment of organophosphate poisoning.

Animals↗

PubMed1995

Formation of N-7-(2-carbamoyl-2-hydroxyethyl)guanine in DNA of the mouse and the rat following intraperitoneal administration of [14C]acrylamide.

Acrylamide is an alkylating agent which reacts very slowly in direct reactions with DNA and is negative in the Ames test, but is carcinogenic in mice and rats. In order to explain the cancer-initiating properties of acrylamide we have studied DNA adduct formation in vitro with a metabolizing system and in vivo in mice and rats following i.p. administration of 14C-labeled acrylamide. A major adduct found in both species was N-7-(2-carbamoyl-2-hydroxy-ethyl)guanine, formed by reaction of the DNA with the epoxide metabolite glycidamide. The levels of this adduct were similar in the different organs of the two rodent species, which supports the notion that glycidamide is relatively evenly distributed among tissues and that the organ-specificity in acrylamide carcinogenesis cannot be explained by a selective accumulation of the DNA-reactive metabolite in target organs.

Acrylamide↗

PubMed1994

Inhibition of muscarinic receptor-stimulated phosphoinositide metabolism by cocaine, norcocaine and cocaethylene in rat brain.

The interaction of cocaine, its metabolites norcocaine and benzoylecgonine, and cocaethylene, which is formed following a combined cocaine and ethanol exposure, with muscarinic receptor binding and phosphoinositide metabolism was investigated in brain from immature rats. Cocaine and norcocaine inhibited binding of [3H]telenzepine and carbachol-stimulated phosphoinositide metabolism in cerebral cortex, while benzoylecgonine was devoid of any inhibitory activity. Cocaethylene was the most potent inhibitor of both binding and phosphoinositide metabolism. The effect of cocaine was more pronounced at the muscarinic receptors, but a small inhibition of histamine--and serotonin--stimulated phosphoinositide metabolism was also observed.

Animals↗

PubMed1994

Developmental neurotoxicity of ethanol: further evidence for an involvement of muscarinic receptor-stimulated phosphoinositide hydrolysis.

Various lines of evidence suggest that muscarinic receptor-stimulated phosphoinositide hydrolysis during postnatal development in the rat brain may play a relevant role in glial cell proliferation and neuronal differentiation. We have previously shown that administration of ethanol to developing rats during the brain growth spurt causes microencephaly and selectively decreases muscarinic receptor-stimulated phosphoinositide hydrolysis. In the present study we have investigated the sensitivity of the phosphoinositide system coupled to muscarinic receptors to ethanol inhibition during distinct stages of the brain growth spurt. Different groups of rats were treated for 3 days with ethanol (4 g/kg per day) on postnatal days 2-4 (initial), 6-8 or 10-12 (peak), 13-15 (final stage of the brain growth spurt). The results show that the period of maximal sensitivity to ethanol of muscarinic receptor-stimulated phosphoinositide hydrolysis coincides with the peak of the brain growth spurt and with the period of maximal efficacy of muscarinic receptor agonists to induce inositol phosphates accumulation. Interestingly, only when muscarinic receptor-stimulated phosphoinositide hydrolysis was inhibited, a significant reduction of brain weight was observed. The close parallel between inhibition of this second messenger response and reduction of brain weight suggests that the phosphoinositide system coupled to muscarinic receptors may represent a target for the neurotoxic effects of ethanol during this stage of brain development.

Animals↗

PubMed1994

Effects of styrene oxide on differentiation and viability of rodent embryo cultures.

Epidemiological investigations on the reproductive and developmental outcomes of rubber and plastic industry workers have focused on the potential developmental toxicity of styrene, an important occupational chemical, and its metabolite, styrene oxide using two in vitro culture systems: micromass cell cultures and whole embryo culture (WEC). Further, we have compared the effects of styrene oxide in both culture systems to the developmental toxicity of styrene in the micromass system. The ability of styrene oxide to affect the differentiation of rat embryo midbrain (CNS) and limb bud (LB) cells was compared to general cytotoxicity over 5 days in high-density micromass cultures. The IC50 for differentiation was 9.2 micrograms/ml (76 microM) for CNS and 6.7 micrograms/ml (56 microM) for LB. The LC50 for cytotoxicity was 9.6 micrograms/ml (80 microM) and 27.5 micrograms/ml (228 microM) for CNS and LB, respectively. The values for CNS sensitivities suggest that inhibition of differentiation is probably a consequence of high levels of cytotoxicity. In contrast, effects of styrene oxide on LB endpoints of differentiation were evident at concentration levels which had minimal effects on cell viability. Styrene alone or in the presence of an exogenous monooxygenase system had minimal effects when tested at concentrations 4-12 times the highest IC50 values seen with styrene oxide alone. In whole embryo culture experiments, styrene oxide produced growth retardation and embryo malformations (primarily neural) with an MC50 value of 20 micrograms/ml (167 microM). The LC50 value for styrene oxide was approximately 1.7-fold higher (33.2 micrograms/ml, 276 microM). Our in vitro studies suggest that further evaluations of the relationship of developmental toxicity and generalized cytotoxicity of styrene and its metabolite are needed, especially given the low concentrations at which effects were seen.

Animals↗

PubMed1994

Platelet monoamine oxidase B activity in workers exposed to styrene.

A cross-sectional study was conducted to evaluate monoamine oxidase type B (MAO-B) activity in platelets as a biomarker of effect of styrene and perchloroethylene exposures. MAO-B is an enzyme system involved in dopamine catabolism, the impairment of which has been postulated as a mechanism of styrene-induced neurotoxicity. We previously observed an inverse association between blood styrene and MAO-B among reinforced plastics manufacturing workers. The present study included 59 male boat plant workers exposed to styrene (exposure range < 1-144 ppm, 8-h TWA). Two comparison groups comprised six male dry cleaning workers exposed to perchloroethylene (PCE; exposure range < 2-37 ppm) and 14 male laundry workers not exposed to either agent. Respiratory protection was not used by any of the styrene- or PCE-exposed workers; thus, air concentrations were regarded as valid exposure indicators. MAO-B activity (pmol/10(8) cells/h) was measured in peripheral blood platelets, using phenylethylamine as substrate. Only small overall mean differences in MAO-B were observed among the three groups; mean values were 4.21, 4.51, and 4.12 for the styrene-exposed, PCE-exposed, and laundry workers, respectively. Despite the absence of gross differences among the groups, styrene exposure was inversely related to MAO-B. Mean values for four increasing exposure group quartiles were: 5.60, 4.13, 3.69, and 3.44. The Spearman rank correlation coefficient for styrene with MAO-B was -0.41. Adjustment for age, medication use, smoking, and alcohol consumption had only a minimal effect on this trend.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗

PubMed1994

Lack of m2 muscarinic acetylcholine receptor mRNA in rat lymphocytes.

The presence of muscarinic acetylcholine receptors on lymphocytes has been demonstrated by radioligand binding experiments. Although the specific subtype(s) of muscarinic acetylcholine receptors expressed in lymphocytes is still unknown, some reports suggest the presence of the m2 subtype. In this study we analyzed the expression of m2 subtype mRNA in rat mononuclear cells, B lymphocytes and T lymphocytes by Northern blot hybridization and reverse transcription-polymerase chain reaction. Positive signals for the presence of m2 mRNA were found in rat heart, brainstem, cerebral cortex, corpus striatum and hippocampus, which were used as positive controls. On the other hand, no expression of m2 was detected in lymphocytes. These results indicate that mRNA for the m2 subtype is absent in rat lymphocytes and that one or more other subtypes may be responsible for the reported results in binding experiments.

Animals↗

PubMed1994

Interaction of organophosphorus compounds with muscarinic receptors in SH-SY5Y human neuroblastoma cells.

Human neuroblastoma cells (line SH-SY5Y) were used to examine the interaction of single exposure to organophosphorus compounds (OPs) with muscarinic receptors. In this study, SH-SY5Y cells were exposed for 30 min to concentrations of paraoxon, diisopropyl phosphorofluoridate (DFP), phenyl saligenin cyclic phosphate (PSP), and mipafox (N,N'-diisopropyl phosphorodiamide fluoridate) that ranged between 10(-9) M and 10(-3) M (10(-2) M for mipafox). Ability to interfere with muscarinic receptor binding was determined by change in the binding of the nonspecific antagonist [3H]-N-methylscopolamine (3H-NMS). Concentrations of paraoxon > 0.5 x 10(-3) M and PSP 1 x 10(-3) M significantly inhibited the binding of a saturating concentration of 3H-NMS. Concentrations of > 10(-5) M paraoxon or PSP could significantly inhibit the binding of a half-saturating concentration of 3H-NMS. Studies using specific antagonists for muscarinic subtypes (pirenzepine for M1, AFDX-116 for M2, and 4-DAMP for M3) indicated that SH-SY5Y cells have muscarinic receptors most sensitive to the specific antagonist for the M3 subtype (IC50 of 10(-8) M for 4-DAMP compared to 2.5 x 10(-6) M and 2.7 x 10(-5) M for pirenzepine and AFDX-116, respectively). As M3 receptor stimulation results in formation of inositol phosphates from membrane phosphoinositides the capability of OPs to alter levels of inositol phosphates and agonist-stimulated increases in inositol phosphate formation was examined. Intact cells were prelabeled with [3H]myo-inositol and then incubated for 15 min with the OPs before addition of 10(-5) M to 10(-3) M carbachol. Levels of inositol phosphates were determined as the amount of aqueous soluble radiolabeled product extracted from the reaction mixture. Paraoxon and PSP, but not mipafox or DFP, decreased basal levels of inositol phosphates in a concentration-related manner. This could be overcome in cells stimulated with carbachol, a muscarinic agonist, and with sodium fluoride, which does not act at muscarinic receptors. These results indicate that certain OPs, upon acute exposure, interact with muscarinic receptors, but that they also have effects on levels of inositol phosphates that may be associated with another site of action in SH-SY5Y cells.

Carbachol↗

PubMed1994

Nutrition and alcohol neurotoxicity.

Neurological complications of alcoholism such as Wernicke-Korsakoff syndrome and polyneuropathy often originate from interactive factors involving direct nervous system toxicity of ethanol and nutrient deficiencies associated to heavy drinking. Not all patients are equally susceptible to these disorders and a genetic predisposition to thiamine deficiency has been described in subjects with Wernicke's encephalopathy. At moderate alcohol dosages, nutrient abnormalities may be marginal, inducing no obvious manifestations until other neurotoxic agents are absorbed. Examples are presented illustrating the interaction of ethanol and styrene on brain glutathione metabolism in rats, and cases of methanol poisoning in alcoholics. In these patients, ethanol-induced folate deficiency can potentiate visual toxicity of methanol due to impairment of the folate-dependent pathway involved in formate detoxication. The notion that nutritional deficiencies and ethanol toxicity may act synergistically in the nervous system outlines the importance of adequate nutritional strategies in the treatment of alcoholism and also indicates that methodological flaws may result during experimental studies from failure to control for nutritional variables.

Animals↗

PubMed1994

Signal transduction mechanisms in developmental neurotoxicity: the phosphoinositide pathway.

The importance of activation of second messenger systems which follows the interaction of neurotransmitters, hormones and growth factors with their receptors is increasingly recognized. The phosphoinositide/protein kinase C pathway has received particular attention in the past decade, particularly for its involvement in the control of intracellular calcium levels. Though new functions of this metabolic pathway in the nervous system are continuously discovered, little is still known about the possible role of phosphoinositide metabolism in mediating various aspects of neurotoxicity. Limited information also exists on various aspects of inositol metabolism during brain development and, as a consequence, on its possible role in developmental neurotoxicity. In this brief review, some of the current concepts of receptor--activated phosphoinositide metabolism, particularly during brain development, are summarized. Two examples are presented to indicate its possible involvement in developmental neurotoxicity. The acetylcholine muscarinic receptor--stimulated inositol metabolism is discussed as a potential target for the developmental neurotoxicity of ethanol. The possible contribution of the glutamate metabotropic receptors in the neurotoxicity of excitatory amino acid in the immature brain is also discussed.

Animals↗

PubMed1993

Interaction of ethanol with muscarinic receptor-stimulated phosphoinositide metabolism during the brain growth spurt in the rat: role of acetaldehyde.

The developing brain is extremely sensitive to the neurotoxicity of ethanol; however, the mechanism(s) of its developmental neurotoxicity are still elusive. In the developing rat brain, ethanol exerts an age-, brain region-, and receptor-specific inhibitory effect on muscarinic receptor-stimulated phosphoinositide metabolism, which may be linked to some of the neurotoxic effects of ethanol found in children with fetal alcohol syndrome. Since some studies have suggested that the ethanol metabolite acetaldehyde may mediate, at least in part, the developmental effects of ethanol, in the present study we have examined whether acetaldehyde would inhibit carbachol-stimulated phosphoinositide metabolism in brain slices from immature rats. We also tested propionaldehyde, the corresponding aldehyde of n-propanol, another alcohol shown to cause microencephaly and to affect phosphoinositide metabolism in the developing rat. Neither acetaldehyde nor propionaldehyde, at concentrations up to 1 mM, had any inhibitory effect on this system, while the two alcohols did, as previously reported. These results suggest that ethanol itself may be the primary agent responsible for its developmental neurotoxicity.

1-Propanol↗

PubMed1993

Interaction of the neurotoxic pesticides ivermectin and lindane with the enteric GABAA receptor-ionophore complex in the guinea-pig.

In isolated segments of guinea-pig small intestine, gamma-aminobutyric acid (GABA) (3-300 microM), the GABAA receptor agonist 3-aminopropane sulphonic acid (3-APS) (3-300 microM) and ivermectin (1-300 microM) caused concentration-dependent nerve-mediated cholinergic contractions sensitive to tetrodotoxin (1 microM) and hyoscine (1 microM). The EC50 values were 30.2 +/- 4.3, 24.6 +/- 3.1 and 4.8 +/- 0.6 microM, respectively. Picrotoxinin (10 microM), an allosteric blocker of the Cl- channel associated with GABAA receptors, non-competitively antagonized the contractile response caused by each agonist. Like picrotoxinin, lindane (10, 30 microM) caused a dose-related shift to the right of the concentration-response curve to GABA, 3-APS and ivermectin with depression of the maximum response. SR 95531 (3 microM), a competitive antagonist of GABAA receptors, caused a parallel dextral shift of the concentration-response curve to ivermectin with an apparent single point pA2 value of 6.5. Our results suggest that ivermectin and lindane, two neurotoxic pesticides interfering with central GABAErgic transmission, exert agonist and non-competitive antagonist properties at the enteric GABAA receptor-ionophore complex. This peripheral complex can thus be considered as an additional target for the action of both these compounds.

Animals↗

PubMed1993

Cyclic GMP formation induced by muscarinic receptors is mediated by nitric oxide synthesis in rat cortical primary cultures.

In rat primary cortical cultures, carbachol caused a time- and concentration-dependent increase in guanosine cyclic 3',5'-monophosphate (cGMP) levels, which was antagonized by the muscarinic antagonist atropine. Glutamate and sodium nitroprusside also caused large increases in cGMP levels, as previously reported. Two nitric oxide (NO) synthase inhibitors, L-NG-nitroarginine and L-NG-monomethylarginine, were tested for their ability to inhibit the carbachol- and the glutamate-induced cGMP formation. The cGMP response to carbachol was decreased by both compounds in a dose-dependent fashion. The effect of L-NG-nitroarginine was competitively reversed by addition of an excess of L-arginine. Similarly, the stimulatory effect of glutamate on cGMP levels was antagonized by L-NG-nitroarginine and L-NG-monomethylarginine. Hemoglobin, a scavenger of NO, also blocked the carbachol-stimulated cGMP production. These results indicate that muscarinic receptor-stimulated cGMP formation in rat cerebral cortex is mediated by NO.

Amino Acid Oxidoreductases↗