Involvement of microglia-neuron interactions in the tumor necrosis factor-alpha release, microglial activation, and neurodegeneration induced by trimethyltin.

Trimethyltin (TMT) is a neurotoxicant known to induce early microglial activation. The present study was undertaken to investigate the role played by these microglial cells in the TMT-induced neurotoxicity. The effects of TMT were investigated in monolayer cultures of isolated microglia or in neuron-enriched cultures and in neuron-microglia and astrocyte-microglia cocultures. The end points used were morphological criteria; evaluation of cell death and cell proliferation; and measurements of tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), and nitric oxide (NO) release in culture supernatant. The results showed that, in cultures of microglia, TMT (10(-6) M) caused, after a 5-day treatment, an increased release of TNF-alpha, without affecting microglial shape or cell viability. When microglia were cocultured with astrocytes, TNF-alpha release was decreased to undetectable levels. In contrast, in neuron-microglia cocultures, TNF-alpha levels were found to increase at lower concentrations of TMT (i.e., 10(-8) M). Moreover, at 10(-6) M of TMT, microglia displayed further morphological activation, as suggested by process retraction and by decrease in cell size. No morphological activation was observed in cultures of isolated microglial cells and in astrocyte-microglia cocultures. With regard to neurons, 10(-6) M of TMT induced about 30% of cell death, when applied to neuron-enriched cultures, whereas close to 100% of neuronal death was observed in neuron-microglia cocultures. In conclusion, whereas astrocytes may rather dampen the microglial activation by decreasing microglial TNF-alpha production, neuronal-microglial interactions lead to enhanced microglial activation. This microglial activation, in turn, exacerbates the neurotoxic effects of TMT. TNF-alpha may play a major role in such cell-cell communications.

Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity.

Activation of microglia is a well-documented phenomenon associated with diverse pathological conditions of the central nervous system. In order to investigate the involvement of microglial cells in the neurotoxic action of the heavy metal compound trimethyltin, three-dimensional brain cell cultures were treated during an early developmental period, using concentrations at or below the limit of cytotoxicity. Microglial cells were studied by cytochemical staining, using horseradish peroxidase-conjugated B4 isolectin (GSI-B4). In parallel, neurotoxic effects were assessed by determining the content of synaptophysin and synapsin I, both in the total homogenates and in the synaptosomal fraction of the cultures. Changes in the content of the specific growth cone protein, GAP-43, were also analyzed. It was found that low, non-cytotoxic concentrations of TMT (10(-9) to 10(-8) M) caused a significant increase in the number and/or the clustering of microglial cells. A decrease in the synaptic protein (synapsin I, synaptophysin) content was detected at 10(-8) M of TMT in synaptosomal fractions, whereas in the total homogenates, changes in synaptic proteins and GAP-43 were observed only at the cytotoxic TMT concentration (10(-6) M). Although it remains to be shown whether the microglial response is caused by direct or indirect action of TMT, the present findings show that microglial responsiveness can be detected prior to any sign of neuronal degeneration, and may serve as a sensitive indicator for heavy metal neurotoxicity in the brain.

Effects of trimethyltin (TMT) on glial and neuronal cells in aggregate cultures: dependence on the developmental stage.

Long-term effects of trimethyltin (TMT) applied at concentrations below the cytotoxic level were examined in three-dimensional cell cultures of fetal rat telencephalon using biochemical, immunochemical and morphological criteria. It was found that in immature cultures low concentrations of TMT (10(-8) M) specifically induced a gliotic response in astrocytes, with increased immunoreactivity for glial fibrillary acidic protein, and a greater number of astrocytic processes. Significant changes in oligodendrocytic and neuronal parameters were found only at 10(-6) M of TMT. In differentiated cultures, distinct changes in cell type-specific parameters occurred at 10(-6) M of TMT (the lowest effective concentration). In addition, different patterns of responses were found for astrocytes and oligodendrocytes, as compared to immature cultures. These results suggest that among neural cells, astroblasts are most sensitive to TMT, and that the glial responses to this neurotoxicant are development-dependent.

Assessment of the astrogliotic responses of three human astrocytoma cell lines to ethanol, trimethyltin chloride and acrylamide

The astrogliotic responses of the CCF-STTG1, U251-MG, and U373-MG human astrocytoma lines were determined after exposure to ethanol, trimethyltin chloride (TMTC), and acrylamide over 4, 16, and 24 h. Basal glial fibrillary acidic protein (GFAP) expression in the U-251MG and U373-MG cells was 10-fold greater than the CCF-STGG1 line. Ethanol treatment over 24 h, but not at 4 and 16 h, resulted in significant increases in GFAP in all three glioma lines at sub-cytotoxic levels; the GFAP responses in the CCF-STTG1 line were the most sensitive, as concentrations of 0.1 and 1 mM led to increases in GFAP expression compared with control of 56.8 ± 15.7 and 58.9 ± 11.5%, respectively (P < 0.05). Treatment with TMTC (1 μM) over 4 h showed elevated GFAP expression in the U251-MG cell line to 28.0 ± 15.7% above control levels (P < 0.01), but not in the other U373-MG or CCF-STTG1 cells. At 4 h, MTT turnover was markedly increased compared with control, particularly in the U373-MG line at concentrations as low as 1 μM (17.1 ± 2.3%; P < 0.01). TMTC exposure over 16 and 24 h resulted in reduction in GFAP expression in all three lines at concentrations; at 24 h incubation, the reduction was >50% (P < 0.01). There were no changes in GFAP expression or MTT turnover in response to acrylamide except at the highest concentration ranges of 10-100 mM. This study underlines the significance of period of exposure, as well as toxin concentration in astrocytoma cellular response to toxic pressure. © 2007 Elsevier Ireland Ltd. All rights reserved.

Neurotoxicity of dibutyltin in aggregating brain cell cultures.

Dibutyltin (DBT) compounds are used primarily as stabilizers for polyvinyl chloride (PVC) plastics. Small quantities can be released from PVC containers into stored liquids. The neurotoxicological potential of DBT was tested in aggregating brain cell cultures after a 10-day treatment with concentrations ranging from 10(-10) to 10(-6)m, either during an early developmental period, or during a phase of advanced maturation. Changes in protein content, DNA labelling and cell type-specific enzyme activities were measured as end points. DBT caused general cytotoxicity at 10(-6)m in both immature and differentiated cultures. At 10(-7)m, it affected the myelin content and the cholinergic neurons in both states of maturation, while GABAergic neurons remained unchanged. Astrocyte and oligodendrocyte markers were diminished at 10(-7)m of DBT exclusively in immature cultures. DBT uptake by undifferentiated and differentiated cells was similar at this concentration. Whereas trimethyltin (TMT) is known to induce gliosis and triethyltin (TET) to cause demyelination and affect GABAergic neurons, DBT appeared to be more toxic than TMT, and to present a distinct toxicological pattern.

Highly efficient, multigram and enantiopure synthesis of (S)-2-(2,4′-bithiazol-2-yl)pyrrolidine

(S)-2-(4-Bromo-2,4"-bithiazole)-1-(tert-butoxycarbonyl)pyrrolidine ((S)-1) was obtained as a single enantiomer and in high yield by means of a two-step modified Hantzsch thiazole synthesis reaction when bromoketone 3 and thioamide (S)-4 were used. Further conversion of (S)-1 into trimethyltin derivative (S)-2 broadens the scope for further cross-coupling reactions.

Unsaturated σ-hydrocarbyl transition-metal complexes. Part 2. Synthesis and reactions of vinylplatinum complexes and a comparison with analogous fluorovinyl and alkynyl complexes

Alkenyl (CHCH2 or CFCF2) or alkynyl (CCPh) derivatives of trimethyltin are shown to be superior to lithium or magnesium reagents for the synthesis of corresponding mono-organoplatinum(II) species by metathesis (L = SnMe3R +cis-[PtCl2L2]→trans-[PtRClL2]+ SnMe3Cl tertiary phosphine). The reactivity order for SnMe3R is R = CCPh > CFCF2 > CHCH2. This order is also found for oxidative addition of SnMe3R to Pt0 to give cis-[PtRL2(SnMe3)]. When the latter complex (R = CHCH2) reacts with X2 or MeX further oxidative addition occurs exclusively at the platinum centre. Aromatic isonitriles (R′NC)co-ordinate to the platinum and give insertion products trans-[Pt{C(CHCH2)= NR′}ClL2] on heating or carbene complexes with NBunH2. The alkynyl trans-[Pt(CCPh)ClL2] also forms 1 :1 adducts with R′NC and carbene complexes therefrom, but no insertion products. Spectroscopic data for the new complexes are presented.

The synthesis, structure, and reactions of some simple unsaturated σ-hydrocarbylplatinum(II) complexes

Trimethyltin compounds Me3SnR(R = CHCH2, CFCF2, or CCPh) are selective reagents for the synthesis of unsaturated hydrocarbyl derivatives such as trans-PtCl(R)(PPhEt2)2, by R/Cl exchange or oxidative addition (e.g., to Pt(PPh3)3); single crystal X-ray analyses of two such compounds (R = CHCH2 or CCPh) show that the trans-influence of R has only a low sensitivity to hybridisation at carbon, with sp3 > sp ⩾ sp2.

Evaluation of the importance of astrocytes when screening for acute toxicity in neuronal cell systems

Reliable, high throughput, in vitro preliminary screening batteries have the potential to greatly accelerate the rate at which regulatory neurotoxicity data is generated. This study evaluated the importance of astrocytes when predicting acute toxic potential using a neuronal screening battery of pure neuronal (NT2.N) and astrocytic (NT2.A) and integrated neuronal/astrocytic (NT2.N/A) cell systems derived from the human NT2.D1 cell line, using biochemical endpoints (mitochondrial membrane potential (MMP) depolarisation and ATP and GSH depletion). Following exposure for 72 h, the known acute human neurotoxicants trimethyltin-chloride, chloroquine and 6-hydroxydopamine were frequently capable of disrupting biochemical processes in all of the cell systems at non-cytotoxic concentrations. Astrocytes provide key metabolic and protective support to neurons during toxic challenge in vivo and generally the astrocyte containing cell systems showed increased tolerance to toxicant insult compared with the NT2.N mono-culture in vitro. Whilst there was no consistent relationship between MMP, ATP and GSH log IC(50) values for the NT2.N/A and NT2.A cell systems, these data did provide preliminary evidence of modulation of the acute neuronal toxic response by astrocytes. In conclusion, the suitability of NT2 neurons and astrocytes as cell systems for acute toxicity screening deserves further investigation.

Development of a post-mitotic, neuronal-astrocytic cell system, for the prediction of acute neurotoxicity in humans

The human NT2.D1 cell line was differentiated to form both a 1:2 co-culture of post-mitotic NT2 neuronal and NT2 astrocytic (NT2.N/A) cells and a pure NT2.N culture. The respective sensitivities to several test chemicals of the NT2.N/A, the NT2.N, and the NT2.D1 cells were evaluated and compared with the CCF-STTG1 astrocytoma cell line, using a combination of basal cytotoxicity and biochemical endpoints. Using the MTT assay, the basal cytotoxicity data estimated the comparative toxicities of the test chemicals (chronic neurotoxin 2,5-hexanedione, cytotoxins 2,3- and 3,4-hexanedione and acute neurotoxins tributyltin- and trimethyltin- chloride) and also provided the non-cytotoxic concentration-range for each compound. Biochemical endpoints examined over the non-cytotoxic range included assays for ATP levels, oxidative status (H2O2 and GSH levels) and caspase-3 levels as an indicator of apoptosis. although the endpoints did not demonstrate the known neurotoxicants to be consistently more toxic to the cell systems with the greatest number of neuronal properties, the NT2 astrocytes appeared to contribute positively to NT2 neuronal health following exposure to all the test chemicals. The NT2.N/A co-culture generally maintained superior ATP and GSH levels and reduced H2O2 levels in comparison with the NT2.N mono-culture. In addition, the pure NT2.N culture showed a significantly lower level of caspase-3 activation compared with the co-culture, suggesting NT2 astrocytes may be important in modulating the mode of cell death following toxic insult. Overall, these studies provide evidence that an in vitro integrated population of post-mitotic human neurons and astrocytes may offer significant relevance to the human in vivo heterogeneous nervous system, when initially screening compounds for acute neurotoxic potential.

Investigation of aspects of reactive gliosis in human astrocytoma cell lines:application for toxicity assessment

The process of astrogliosis, or reactive gliosis, is a typical response of astrocytes to a wide range of physical and chemical injuries. The up-regulation of the astrocyte specific glial fibrillary acidic protein (GFAP) is a hallmark of reactive gliosis and is widely used as a marker to identify the response. In order to develop a reliable, sensitive and high throughput astrocyte toxicity assay that is more relevant to the human response than existing animal cell based models, the U251-MG, U373-MG and CCF-STTG 1 human astrocytoma cell lines were investigated for their ability to exhibit reactive-like changes following exposure to ethanol, chloroquine diphosphate, trimethyltin chloride and acrylamide. Cytotoxicity analysis showed that the astrocytic cells were generally more resistant to the cytotoxic effects of the agents than the SH-SY5Y neuroblastoma cells. Retinoic acid induced differentiation of the SH-SY5Y line was also seen to confer some degree of resistance to toxicant exposure, particularly in the case of ethanol. Using a cell based ELISA for GFAP together with concurrent assays for metabolic activity and cell number, each of the three cell lines responded to toxicant exposure by an increase in GFAP immunoreactivity (GFAP-IR), or by increased metabolic activity. Ethanol, chloroquine diphosphate, trimethyltin chloride and bacterial lipopolysaccharide all induced either GFAP or MTT increases depending upon the cell line, dose and exposure time. Preliminary investigations of additional aspects of astrocytic injury indicated that IL-6, but not TNF-α. or nitric oxide, is released following exposure to each of the compounds, with the exception of acrylamide. It is clear that these human astrocytoma cell lines are capable of responding to toxicant exposure in a manner typical of reactive gliosis and are therefore a valuable cellular model in the assessment of in vitro neurotoxicity.

Further preliminary assessment of three human glioma cell lines as models of human astrocytic toxicity in vitro

Three human astroglioma lines U251-MG, U373-MG and CCF-STTG1 have been evaluated further as possible models for astrocytotoxicity (GFAP and IL-6 release). The effects of bacterial lipopolysaccharide, chloroquine diphosphate and acrylamide were studied on GFAP expression and LPS, chloroquine diphosphate, ethanol, trimethyltin chloride (TMTC) and acrylamide were examined on interleukin-6 (IL-6) release in the U373-MG line only. At 4-h LIPS elevated GFAP (17.0±5.0% P < 0.05) above control in the U251-MG cell line only. Chloroquine diphosphate over 4 h in the U251-MG line resulted in an increase in GFAP-IR to 20.3 ±4.2% and 21.1 ± 4.1 % above control levels 0.1 µM (P< 0.05) and 1 µM (P< 0.05) respectively. CQD was associated with decreases in MTT turnover, particularly after 24 h incubation. With the U373-MG line, LPS (0.5 µg/ml) increased IL-6 expression 640% above control (P < 0.001), whilst chloroquine diphosphate (100 µM), ethanol (10mM) and TMTC chloride (1 µM) also increased IL-6. It is possible that batteries of astrocytic human glioma cell lines may be applicable to the sensitive evaluation of toxicants on astrogliotic expression markers such as GFAP and IL-6.