Pilar Sanz

Comparative in vitro effects of sodium arsenite and sodium arsenate on neuroblastoma cells

The toxic effects of arsenic at different cellular levels were assessed using two inorganic chemical species: sodium arsenite and sodium arsenate, representing the trivalent and pentavalent states of arsenic, respectively. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed for 24 h, and cytotoxic effects evaluated were: cell proliferation by quantification of total protein content; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage; lysosomal hexosaminidase release; lactate dehydrogenase activity; mitochondrial succinate dehydrogenase activity; relative neutral red uptake by lysosomes; lysosomal hexosaminidase sphingolipid degradation activity; and acetylcholinesterase activity. As(III) was found to be five times more toxic than As(V) to neuroblastoma cell proliferation, but the relative extent of other alterations differed. Special sensitivity was detected for lactate dehydrogenase inhibition. Hexosaminidase activity was also very susceptible, being inhibited at low concentrations and stimulated at high concentrations. Less sensitive were the inhibition of cell proliferation, relative neutral red uptake, and acetylcholinesterase activity. As(III) was lysosomotropic, with secretion of hexosaminidase, but the release was decreased by As(V). Mitochondrial succinate dehydrogenase was inhibited by As(III) and stimulated by As(V). Minor sensitivity to cytoplasmic lactate dehydrogenase leakage for both compounds also shows that functional metabolic alterations produced by arsenic are more important than structural damage.

In vitro effects of mercuric chloride and methylmercury chloride on neuroblastoma cells

An in vitro model system has been developed to establish dose-response relationships of mercuric chloride (HgCl(2)) and methylmercuric chloride (HgCH(3)Cl). Mouse neuroblastoma cell cultures (Neuro-2a) were exposed for 24 hr and cytotoxic effects evaluated with eight different endpoints. Toxic indicators assessed in the in vitro test system were as follows: cell proliferation by quantification of total protein content; cytoplasmic membrane integrity by cytosolic lactate dehydrogenase leakage; lysosomal membrane stability by hexosaminidase release; lactate dehydrogenase activity; mitochondrial succinate dehydrogenase activity; relative neutral red uptake by lysosomes; lysosomal hexosaminidase sphingolipid degradation activity; acetylcholinesterase activity. The toxicity of the two chemical species of mercury on neuroblastoma cells differed. HgCl(2) inhibited LDH activity specifically and very potently. Gross disruption of cytoplasmic membrane was accompanied by stimulation of hexosaminidase. HgCH(3)Cl was 50 times more toxic than HgCl(2) to cell proliferation and also caused important alterations in both membrane stability and metabolic activities over a narrow range of doses. The data suggest that HgCl(2) acts mainly on cell membranes and LDH, whereas, although HgCH(3)Cl is more cytotoxic, it does not affect any of the above-mentioned endpoints as specifically.

Red Blood Cell and Total Blood Acetylcholinesterase and Plasma Pseudocholinesterase In Humans: Observed Variances

Although acetylcholinesterase is the target molecule of organophosphate poisoning, it is not always assayed in clinical evaluations which include only the determination of plasma or serum cholinesterase. In this paper we present observations on workers exposed to, or poisoned by, ethylparathion. Acetylcholinesterase decreased earlier and more intensely than cholinesterase, with the suggestion of an initial increase of acetylcholinesterase activity in newly exposed, workers. A simplified standard Ellman assay of total acetylcholinesterase activity of hemolyzed total blood correlated with that of washed erythrocyte acetylcholinesterase. All results were standardized to both red blood cell and hemoglobin concentration. Normal values in a group of unexposed subjects were acetylcholinesterase: 1225 +/- 181 nU x 10/RBC and 39.30 +/- 5.05 U/g Hb for men, 1321 +/- 234 nU x 10/RBC and 42.57 +/- 6.85 U/g Hb for women. Differences between total and erythrocyte acetylcholinesterase were not statistically significant. Plasma cholinesterase appeared to be decreased in pregnancy and increased in anesthesia, liver and kidney disease and neuropathologic conditions attributed to metal poisoning while total acetylcholinesterase was unaffected. The determination of both cholinesterase and acetylcholinesterase assists the evaluation of individuals exposed to or poisoned by organophosphate, the differentiation of other conditions affecting cholinesterase and the recognition of genetically atypical cholinesterase.

In vitro effects of lithium and nickel at different levels on Neuro-2a mouse Neuroblastoma cells

Lithium and nickel present low toxicity, but are able to cause alterations in different tissues. The toxic effects of lithium and nickel at different cellular levels were assessed using two inorganic chemical species: lithium chloride and nickel(II) chloride. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed to both compounds for 24 h. The cytotoxic effects evaluated were cell proliferation by quantification of total protein content, cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage, and lysosomal hexosaminidase release. Metabolic markers were lactate dehydrogenase activity and mitochondrial succinate dehydrogenase activity. Lysosomal markers were relative neutral red uptake by lysosomes, and lysosomal hexosaminidase sphingolipid degradation activity. Acetylcholinesterase activity on intact cells was also quantified. Nickel was found to be 36 times more toxic than lithium to neuroblastoma cell proliferation (EC(50)= 0.29 and 10.5 mM, respectively), but the relative extent of other alterations differed. Lithium stimulated nearly all the indicators studied, particularly lactate dehydrogenase, mitochondrial succinate dehydrogenase and acetylcholinesterase activities, as well as hexosaminidase release. In contrast, nickel mainly stimulated hexosaminidase release and inhibited lactate dehydrogenase activity. The stabilization of the cytoplasmic membrane to lactate dehydrogenase leakage simultaneously with the secretion of lysosomal hexosaminidase for both compounds also shows that functional metabolic alterations produced by lithium and nickel are more important than cytoplasmic damage.

In vitro effects of thallium on mouse neuroblastoma cells

To compare the effects of thallium at different cellular levels, cultured mouse neuroblastoma cells (Neuro-2A) were exposed for 24 hr to thallium(I) acetate. The following toxic indicators were assessed in the in vitro test system: cell proliferation by quantification of total protein content of the culture; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase (LDH) leakage; lysosomal hexosaminidase release; lactate dehydrogenase activity; mitochondrial succinate dehydrogenase activity; relative neutral red uptake by lysosomes; lysosomal hexosaminidase sphingolipid degradation activity; acetylcholinesterase activity. The effects of thallium on the various indicators differed. Neural acetylcholinesterase activity was extremely sensitive to T1 inhibition. In contrast, hexosaminidase, an enzyme involved in glycosphingolipid degradation, was stimulated prior to cytoplasmic membrane disruption detected as LDH leakage. Relative neutral red uptake was slightly more sensitive than cell growth inhibition and the reduction in hexosaminidase release suggests an interaction with lysosomes. The low degree of sensitivity of cell proliferation, as judged by the protein content of the cultures, may reflect inhibition of protein degradation. LDH glycolytic activity was severely inhibited, but succinate dehydrogenase activity in the citric acid cycle was increased, probably owing to the mitochondrial accumulation of thallium.

Comparative effects of the metabolic inhibitors 2,4-dinitrophenol and iodoacetate on mouse neuroblastoma cells in vitro

The toxic effects of two metabolic inhibitors, dinitrophenol and iodoacetic acid, were compared. Mouse neuroblastoma cell cultures (Neuro-2a) were exposed to different concentrations of the toxic compounds for 24, 48 and 72 h to study basal toxicity effects (cell proliferation by quantification of total protein content (PR) and relative neutral red uptake (RNRU) by lysosomes). The following biochemical indicators assessed in the in vitro test system were: cytosolic phosphofructokinase (PFK) and enolase (ENL) activities in glycolysis; mitochondrial succinate dehydrogenase (SDH) activity in the citric acid cycle; lysosomal beta-galactosidase (GAL) activity; and neuronal acetylcholinesterase (AChE) activity. The effects of the two metabolic inhibitors on the various indicators differed. Iodoacetic acid was found to be far more toxic than dinitrophenol to neuroblastoma cell proliferation at 24 h exposure. Though 2,4-dinitrophenol and iodoacetic acid both inhibited cell proliferation of the neuroblastoma cells, their effects on the other endpoints were opposite. Dinitrophenol was a general activator of the metabolism, particularly affecting lysosomal function. Iodoacetic acid did not significantly alter general metabolism, but considerably modified lysosomal function and AChE activity. The modification of lysosomal function of Neuro-2a cells by the two compounds was quite different: dinitrophenol increased RNRU and GAL activity, and iodoacetic acid decreased both parameters.

Morphological, biochemical and molecular effects of cocaine on mouse neuroblastoma cells culture in vitro

In order to compare the effects of cocaine at morphological, basal cytotoxicity, biochemical and molecular levels, cultured mouse neuroblastoma cells (Neuro-2a) were exposed to a range of concentrations of cocaine hydrochloride. Neuroblastoma cell proliferation, evaluated by quantification of total protein content, was very sensitive to cocaine, being increasingly inhibited from 12 to 72 hr of exposure (EC(50) = 3.1 mm at 24 hr). Cytoplasmic membrane permeability to lactate dehydrogenase was not particularly increased and lysosomal function was stimulated from 0.05 to 1.5 mm, and inhibited from 2.5 mm. A shift to anaerobiosis was detected as intracellular lactate dehydrogenase (LDH) activity was increased and mitochondrial succinate dehydrogenase (SDH) activity decreased. Hexosaminidase (HEX), a lysosomal enzyme involved in sphingolipid degradation, was stimulated only at 1 mm and neural acetylcholinesterase (AChE) activity was stimulated from 2.5 mm. Morphological examination of exposed cultures revealed that most cells became bipolar and multipolar neurons by extension of neurites, but also suffered cytoplasmic vacuolization, hydropic degeneration and nuclear pyknosis. Although cells developing apoptosis were observed, no DNA oligonucleosomal fragmentation was detected by agarose gel electrophoresis of DNA from cells exposed to cocaine. In conclusion, in addition to predominance of anaerobiosis, little disruption of membranes and severe morphologic injury, biochemical and morphological differentiation-like effects were the most prominent alterations produced by cocaine on mouse neuroblastoma cells.

In vitro quantitative structure-activity relationship assessment of pyrrole adducts production by γ-diketone-forming neurotoxic solvents

Organic solvents that are metabolically transformable into gamma-diketones produce central- peripheral distal axonopathy. One of the mechanisms proposed to explain the development of this neuropathy has been the formation of pyrrole adducts between gamma-diketone metabolites and -amine groups of lysine residues on the neuronal cytoskeletal proteins. In vivo studies on the neurotoxic capability of different solvents, derivatives of n-hexane and n-heptane, have previously established the quantitative structure-activity sequence. An in vitro assay for the quantification of pyrrole adduct formation is reported here that allows prediction of neurotoxic potency, using an index of neurotoxic potential. The kinetics of pyrrole adduct formation was established by incubation of each solvent with a purified microsomal fraction of liver from rats preinduced with phenobarbital. The solvents assayed in the in vitro system were 2-hexanone, 3,4-dimethylhexane, 2,5-hexanedione, 3,4-dimethyl-2,5-hexanedione, 2-hexanol and 2,5-hex- anediol as derivatives of n-hexane; and 4-heptanone, 5-methyl-3-heptanone, 6-methyl-2,4-heptanedione and 4-heptanol as derivatives of n-heptane. The results indicate good correlation between neurotoxic potency in vivo and quantitative production of adducts in vitro with both n-hexane and n-heptane derivatives.

Effects of cobalt on mouse neuroblastoma cells cultured in vitro.

To compare the effects of cobalt at different cellular levels, cultured mouse neuroblastoma cells (Neuro-2a) were exposed for 24 hr to cobalt(II) chloride. The following toxicity indicators were assessed: cell proliferation by quantification of total protein content; cytoplasmic membrane integrity to cytosolic lactate dehydrogenase leakage; lysosomal hexosaminidase release; lactate dehydrogenase activity (LDH); mitochondrial succinate dehydrogenase activity (SDH); relative neutral red uptake by lysosomes (RNRU); lysosomal hexosaminidase activity (HEX), and acetylcholinesterase activity (AChE). The effect of cobalt on the various indicators differed. Cobalt was not very toxic to neuroblastoma cell proliferation (EC(50) = 200 mum). Cytoplasmic membrane permeability was not specifically increased, and LDH leakage occurred only at high concentrations, prior to the stimulation of HEX activity, an enzyme involved in sphingolipid degradation. In contrast, cobalt was lysosomotropic, with HEX release. The effects on lysosomal function were also studied with the RNRU, showing stimulation at low concentrations and inhibition at high concentrations. Neural AChE was decreased after an initial stimulation at low concentrations. LDH and SDH intracellular activities were both stimulated from low concentrations, mitochondrial SDH activity being the most sensitive marker studied. Metabolic stimulatory effects induced by cobalt were, therefore, more marked than changes in cytoplasmic and lysosomal membrane permeability.