Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis.

Apoptosis and necrosis are considered conceptually and morphologically distinct forms of cell death. Here, we report that demise of human T cells caused by two classic apoptotic triggers (staurosporin and CD95 stimulation) changed from apoptosis to necrosis, when cells were preemptied of adenosine triphosphate (ATP). Nuclear condensation and DNA fragmentation did not occur in cells predepleted of ATP and treated with either of the two inducers, although the kinetics of cell death were unchanged. Selective and graded repletion of the extramitochondrial ATP/pool with glucose prevented necrosis and restored the ability of the cells to undergo apoptosis. Pulsed ATP/depletion/repletion experiments also showed that ATP generation either by glycolysis or by mitochondria was required for the active execution of the final phase of apoptosis, which involves nuclear condensation and DNA degradation.

Comparative HPLC and ELISA studies for CDT isoform characterization in subjects with alcohol related problems. Prospective application in workplace risk-prevention policy.

Serum carbohydrate-deficient transferrin (CDT) is the most specific marker of chronic alcohol abuse so far. The performance of commercial HPLC over the ELISA method for measurement of CDT was evaluated on a series of 105 serum samples obtained from subjects referred to the Toxicology Laboratory of Salvatore Maugeri Hospital for alcohol-related problems. Compared to ELISA, HPLC analysis was more valuable for determining alcohol-related patterns of CDT isoforms and quantifying serum levels of disialotransferrin that better reflect chronic heavy drinking. Other significant advantages of the HPLC method included reproducible separation and easier detection of glycoform types and genetic transferrin variants that are known to cause falsely high or low results in sera examined by immunoassay. Current scientific evidence indicates that disialotransferrin is the target analyte for CDT determination and HPLC the current CDT analysis reference method. Systematic studies for early assessment of excessive alcohol intake or abuse of alcoholic substances in workers are recommended by the Italian legislation in accordance with the European Alcohol Action Plan (EAAP) launched by the WHO Regional Committee for Europe. These studies are advisable given their potential role in preventing negative effects of alcohol abuse in workplace. A research strategy combining CDT and other laboratory markers with questionnaire and physician interview is recommended for examining subjects with alcohol related problems and the diagnosis of alcoholism. This approach can be applied for alcohol abuse in workplace surveillance.

Analysis of oxidative stress in SK-N-MC neurons exposed to styrene-7,8-oxide.

Styrene-7,8-oxide (SO) is the main metabolite of styrene, a neurotoxic volatile organic compound used industrially. Here we report the novel observation that several markers of oxidative stress were affected in SK-N-MC cells exposed for 16 h to concentrations of SO that induce apoptotic cell death. The production of Thiobarbituric Acid Reactive Substances (TBARS), rose from 69.1 +/- 15.7 nmol/g protein (control) to 119.3 +/- 39.2 and 102.0 +/- 17.3 nmol/g protein after exposure to 0.3 and 1 mM SO, respectively. Carbonyl group levels were significantly enhanced by SO at both concentrations. The lower dose also decreased sulphydryl groups. SO caused a marked oxidative DNA damage, as shown by a fivefold increase in 8-hydroxy-2(')-deoxyguanosine (8-OHdG). In addition, SO exposure resulted in alterations of scavenging abilities, given the reduction of both glutathione (GSH) and glutathione-S-transferase (GST) activity. Induction of expression of the oxidative stress response gene heme-oxygenase-1 (HO-1) and an increased HO-1 activity were also observed. These data provide compelling evidence that oxidative stress significantly contributes to SO toxicity in neuronal cells.

Comparison of in vitro drug-sensitivity of human granulocyte-macrophage progenitors from two different origins: umbilical cord blood and bone marrow.

Predictive in vitro hematotoxicity assays using human cells will provide estimation of tolerable level and aid considerably the development of agents with greater therapeutic activity and less toxicity. Human hematopoietic cells can be derived from three sources: human bone marrow by sternal or femoral aspiration, mobilized peripheral blood, or umbilical cord blood samples collected from placentas after deliveries. Because of the difficulties to have a continuous supply of bone marrow cells from normal human donors and the related ethical problems, we performed a study to compare the sensitivity of human bone marrow cells (h-BMC) and human cord blood cells (h-CBC) to chemicals in order to confirm if h-CBC can readily replace bone marrow cells in checking the sensitivity of GM-CFU progenitors to drugs as preliminarily reported in literature. Our results showed that the prediction of IC50 values in human model is quite similar by using h-BMC or h-CBC. On the contrary, the type of medium influenced in a significant way the ICs determination of some drugs.

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.

Characterization of the 5-HT receptor potentiating neuromuscular cholinergic transmission in strips of human isolated detrusor muscle.

In human isolated detrusor strips, submaximal contractile responses evoked by electrical stimulation were resistant to hexamethonium (30 microM) and abolished by tetrodotoxin (0.6 microM) and hyoscine (1 microM), indicating the activation of postganglionic cholinergic nerves. In methysergide (1 microM) and ondansetron (3 microM) pretreated tissues, 5-hydroxytryptamine (5-HT) (0.3 nM-1 microM) caused a concentration-dependent increase in the amplitude of contractions (pEC50 = 8.1), which was antagonized by the selective 5-HT4 receptor antagonist GR 113808 (3, 10 and 30 nM) in a competitive manner. Schild analysis yielded a pA2 estimate of 8.9, a value comparable to that reported for GR 113808 in other animal and human peripheral tissues (8.8-9.7). Our findings indicate that neuromuscular cholinergic transmission in human isolated detrusor muscle is facilitated by neural 5-HT receptors belonging to the 5-HT4 subtype. The human urinary bladder can thus be regarded as an additional site in which 5-HT4 receptors are distributed.

In vitro myelotoxicity of propanil and 3,4-dichloroaniline on murine and human CFU-E/BFU-E progenitors.

Because of the wide use of pesticides for domestic and industrial purposes, the evaluation of their potential effects is of major concern for public health. The myelotoxicity of the herbicide propanil (3,4-dichloroproprioanilide) and its metabolite 3,4-dichloroaniline (DCA) is well documented in mice, but evidence that pesticides may severely compromise hematopoiesis in humans is lacking. In this study, an interspecies comparison of in vitro toxicity of these two compounds on murine and human burst- and colony-forming unit-erythrocyte (BFU-E, CFU-E) and colony-forming unit-granulocyte/macrophage (CFU-GM) progenitors, has been carried out. Murine bone marrow progenitors and human cord blood cells were exposed to propanil or DCA in doses ranging from 10 micro M to 1000 micro M, and the toxic effect was detected by a clonogenic assay with continuous exposure to the compounds. The results on murine cells indicate that the erythrocytic lineage is the most sensitive target for propanil and DCA. On the other hand, human progenitors seem to be less sensitive to the toxic effects of both compounds than murine progenitors at the same concentrations (IC(50) values are 305.2 +/- 22.6 micro M [total erythroid colonies] and >500 micro M [CFU-GM] for propanil). Propanil was significantly more toxic to human erythroid progenitors than to human CFU-GM progenitors, as was found for the murine cells, emphasizing the role of the heme pathway as the target for propanil. These data confirm the evidence that the compounds investigated interfere with erythroid colony formation at different stages of the differentiation pathway and have different effects according to the dose.

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.

Ethanol selectively interferes with the trophic action of NMDA and carbachol on cultured cerebellar granule neurons undergoing apoptosis.

Exposure of mature rat cerebellar granule neurons to non-depolarizing conditions (5 mM K+) for 24 h resulted in the onset of apoptosis. NMDA, forskolin, carbachol and GABA attenuated low K+-induced toxicity, although to a different extent, with NMDA and GABA being the most effective agents. When cells were co-exposed for 24 h to ethanol, the survival promoting action of NMDA and carbachol, but not that of forskolin and GABA, was attenuated. By contrast, a 24 h cell pre-treatment with ethanol, followed by its removal prior to K+ deprivation, was ineffective towards the neurotrophic action of NMDA and carbachol. The concomitant presence of alcohol and neurotrophic factors was not required for the pro-apoptotic effect of ethanol to be manifest after a long-term alcohol exposure: inhibition of NMDA- and carbachol-mediated neurotrophism was still observed when cells were pre-exposed for 72 h to alcohol in depolarizing conditions, prior to the challenge with 5 mM K+-containing medium and the test compounds in the absence of ethanol. The present study shows that ethanol promotes apoptotic cell death of cultured cerebellar neurons by selectively inhibiting the neurotrophic effect of NMDA and carbachol, and suggests that alcohol may cause permanent changes in the control mechanisms of apoptosis: this finding may have significant implications for the in vivo toxicity of prenatal ethanol exposure on the developing cerebellum.

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.

Neurotoxicity and molecular effects of methylmercury.

The neurotoxicity of high levels of methylmercury (MeHg) and the high susceptibility of the developing brain are well established both in humans and experimental animals. Prenatally poisoned children display a range of effects varying from severe cerebral palsy to subtle developmental delays. Still unknown is the lowest dose that impairs neurodevelopment. The primary source of human exposure is the fish. The data obtained so far from epidemiological studies on fish-eating populations are not consistent. A reference dose of 0.1 microg MeHg/kg per day has been established by the U.S. Environmental Protection Agency based on a study on Iraqi children exposed to MeHg in utero. However, these exposures occurred at high level for a limited period of time, and consequently were not typical of lower chronic exposure levels associated with fish consumption. Major obstacles for estimation of a threshold dose for MeHg include the delayed appearance of the neurodevelopmental effects following prenatal exposure and limited knowledge of cellular and molecular processes underlying these neurological changes. In this respect, a strategy which aims at identifying sensitive molecular targets of MeHg at environmentally relevant levels may prove particularly useful to risk assessment. Here some examples of MeHg molecular effects occurring at low doses/concentrations are presented.

Interaction of aluminum ions with phosphoinositide metabolism in rat cerebral cortical membranes.

Aluminum (Al) is believed to exert a primary role in the neurotoxicity associated with dialysis encephalopathy and has been suggested to be involved in a number of other neurological disorders, including Alzheimer's disease. Al, complexed with fluoride to form fluoroaluminate (AlF4-), can activate the GTP-binding (G) proteins of the adenylate cyclase and retinal cyclic GMP phosphodiesterase systems. Since an involvement of G-proteins with cerebral phosphoinositide (PtdIns) metabolism has also been suggested, in this study we investigated the interaction of the stable GTP analogue GTP(S), Al salts and NaF with this system. In rat cerebral cortical membranes, GTP(S) dose-dependently stimulated [3H]inositol phosphates ([3H]InsPs) accumulation. This effect was potentiated by carbachol and was partially prevented by the GTP-binding antagonist GDP(S), indicating that CNS muscarinic receptor activation is coupled to PtdIns hydrolysis via putative G-protein(s). GTP(S) stimulation was also inhibited by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, which is known to exert a negative feedback control on agonist-stimulated PtdIns metabolism. Both Al salts and NaF mimicked the action of GTP(S) in stimulating PtdIns turnover. Their actions were highly synergistic, suggesting that AlF4- could be the active stimulatory species. However, the stimulatory effects of AlCl3 and/or NaF were not potentiated by carbachol and were not inhibited by GDP(S) and PMA, suggesting that separate sites of action might exist for GTP(S) and AlF4-. In the nervous tissue, activation of PtdIns hydrolysis by Al (probably as AlF4-) may be mediated by activating a regulatory G-protein at a location distinct from the GTP-binding site or by a direct stimulation of phospholipase C.

Interaction of mercury compounds with muscarinic receptor subtypes in the rat brain.

The in vitro effects of mercuric chloride (HgCl2) and methylmercury (CH3HgOH) on the M1 and M2 muscarinic receptor subtypes were investigated in rat brain cortical membranes. HgCl2 and CH3HgOH were almost equipotent in inhibiting the binding of [3H]telenzepine to M1 receptors (IC50s = 2.2 and 3.4 microM, respectively). Conversely, HgCl2 was a thirty-fold more potent inhibitor of [3H]AF-DX 384 binding to M2 sites than CH3HgOH (lC50s = 5 and 149 microM, respectively). In all cases HgCl2 showed steep and monophasic inhibition curves, whereas those of CH3HgOH were biphasic (M1) or shallow (M2). CH3HgOH-induced inhibition of both [3H]telenzepine and [3H]AF-DX 384 binding was of the competitive type, while HgCl2 caused a pronounced reduction of the Bmax value associated with a small change in affinity. CH3HgOH also decreased the affinity of the agonist carbachol for M1 and M2 receptors, while inorganic mercury had minimal effects on the carbachol dose-response curves. These results indicate that inorganic and organic mercury differ in their interaction with muscarinic receptor subtypes and that M1 receptors may represent a preferential target for their effects.

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 in environmental medicine: alterations of cell signalling as early indicators of neurotoxicity.

Many environmental and occupational chemicals are known to affect the central and/or peripheral nervous system, causing changes that may result in neurological and psychiatric disorders. Because of the limited accessibility of the mammalian nervous tissue, new strategies are being developed to identify biochemical parameters of neuronal cell function, which can be measured in easily obtained tissues, such as blood cells, as potential markers of the chemically-induced alterations occurring in the nervous system. This review includes a comparative analysis of the effects of mercurials on calcium signalling in the neuroadrenergic PC12 cells and rat splenic T lymphocytes in an attempt to characterize this second messenger system as a potential indicator of subclinical toxicity. The suitability of neurotransmitter receptors in blood cells, such as the sigma binding sites, as biological markers of psychiatric disorders is also discussed.

[Toxicity of chemical elements]. / Aspetti della tossicità da elementi chimici.

A survey is presented of the most important facets of toxicity due to chemical elements as well as of the mechanisms through which it may be triggered. In particular, a detailed discussion is made on the characteristics shown by arsenic, cadmium, chromium, lead, mercury, barium and beryllium, with specific reference to the influence exerted by physiological, environmental and life-style factors.

Comparative in vitro and ex-vivo myelotoxicity of aflatoxins B1 and M1 on haematopoietic progenitors (BFU-E, CFU-E, and CFU-GM): species-related susceptibility.

Haemato- and myelotoxicity are adverse effects caused by mycotoxins. Due to the relevance of aflatoxins to human health, the present study, employing CFU-GM-, BFU-E- and CFU-E-clonogenic assays, aimed at (i) comparing, in vitro, the sensitivity of human vs. murine haematopoietic progenitors to AFB1 and AFM1 (0.001-50microg/ml), (ii) assessing whether a single AFB1 in vivo treatment (0.3-3mg/kgb.w.) alters the ability of murine bone marrow cells to form myeloid and erythroid colonies, and (iii) comparing the in vitro with the in vitro ex-vivo data. We demonstrated (i) species-related sensitivity to AFB1, showing higher susceptibility of human myeloid and erythroid progenitors (IC(50) values: about 4 times lower in human than in murine cells), (ii) higher sensitivity of CFU-GM and BFU-E colonies, both more markedly affected, particularly by AFB1 (IC(50): 2.45+/-1.08 and 1.82+/-0.8microM for humans, and 11.08+/-2.92 and 1.81+/-0.20microM for mice, respectively), than the mature CFU-E (AFB1 IC(50): 12.58+/-5.4 and 40.27+/-6.05microM), irrespectively of animal species, (iii) regarding AFM1, a species- and lineage-related susceptibility similar to that observed for AFB1 and (iv) lack of effects after AFB1 in vivo treatment on the proliferation of haematopoietic colonies.

Effects of the muscarinic agonist oxotremorine on membrane fluidity in rat lymphocytes.

The muscarinic agonist oxotremorine produced a concentration-dependent increase in membrane fluidity in intact viable rat splenic lymphocytes in vitro. This effect was antagonized by atropine, but only at high concentrations (1 mM), while scopolamine was ineffective. Two other muscarinic agonists, carbachol and pilocarpine, did not affect membrane fluidity in lymphocytes. The fluidizing effect of oxotremorine occurred at both 10 and 37 degrees C with a similar time-course. Oxotremorine also increased membrane fluidity in liposomes of DMPC in gel phase, although its effect was less pronounced than in lymphocytes. The data suggest that the fluidization caused by oxotremorine is primarily nonreceptor-mediated and associated with a nonspecific physicochemical effect.

[Oncogenes as indicators of occupational exposure to carcinogenic agents]. / Oncogeni come indicatori di esposizione professionale ad agenti cancerogeni.

Oncogenes are a class of genes capable of inducing neoplastic alterations of the cell. Recently, oncogenes have received greater attention in environmental medicine, since they may be useful tools for monitoring individuals exposed to carcinogenic chemicals. Their potential use as biomarkers is supported by studies indicating changes in the expression of growth factors and oncoproteins in chemically induced cancer. These alterations may even take place prior to clinical diagnosis. In addition, availability of new immunoblotting techniques, which allow the detection of oncoproteins and growth factors in easily accessible tissues, such as serum and urine, has provided promising preliminary results in humans. In this paper, mechanisms of oncogene activation and their role in occupational carcinogenesis are briefly discussed. Recent human studies suggesting a link between occupational toxicants and oncogenes, as well as a potential use of oncogenes as biomarkers, are also reported. Finally, advantages and limits of oncogenes over more traditional biomarkers are discussed.