• Energy Research

The present Technical Report describes the results obtained in the second and conclusive part of the activity of the reserch contract N. 140053552 ENEL/CNR-ICCOM

The present Technical Report describes the results obtained in the second and conclusive part of the activity of the reserch contract N. 140053552 ENEL/CNR-ICCOM

The heavy metal thallium is an emerging pollutant among the most potentially toxic species to which human populations are exposed. Its harmful effects on living organisms are well-known at high doses, typical of acute intoxication. Its harmful effects at low doses are by far less known. In a previous paper, we reported a TlCl-induced metabolic shift to lactate and ethanol production in living hippocampal HN9.10e neurons that appeared after a single short exposure (48 h) at low doses (1-100 μg/L). This metabolic shift to lactate and ethanol suggests a marked impairment of cell bioenergetics. In this work, we provide detailed evidence for TlCl-induced changes of neuronal morphology and mitochondrial activity. Confocal microscopy and fluorescent probes were used to qualitatively and quantitatively analyze, at the subcellular level, living HN9.10e neurons during and after TlCl exposure. An early onset mitochondrial dysfunction appeared, associated with signs of cellular deregulation such as neurite shortening, loss of substrate adhesion, and increase of cytoplasmic calcium. The dose-dependent alteration of mitochondrial ROS (mtROS) level and of transmembrane mitochondrial potential (ΔΨm) has been observed also for very low TlCl doses (1 μg/L). The treatment with the ATP synthase inhibitor oligomycin revealed a severe impairment of the mitochondrial function, more significant than that measured by the simple quantification of the tetramethylrhodamine methyl ester (TMRM) fluorescence. These results highlight that mitochondria are a key subcellular target of TlCl neurotoxicity. The transmembrane mitochondrial potential was significantly correlated with the ethanol concentration in cell culture medium ( P < 0.001, r = -0.817), suggesting that ethanol could be potentially used as a biomarker of mitochondrial impairment.

The heavy metal thallium is an emerging pollutant among the most potentially toxic species to which human populations are exposed. Its harmful effects on living organisms are well-known at high doses, typical of acute intoxication. Its harmful effects at low doses are by far less known. In a previous paper, we reported a TlCl-induced metabolic shift to lactate and ethanol production in living hippocampal HN9.10e neurons that appeared after a single short exposure (48 h) at low doses (1-100 μg/L). This metabolic shift to lactate and ethanol suggests a marked impairment of cell bioenergetics. In this work, we provide detailed evidence for TlCl-induced changes of neuronal morphology and mitochondrial activity. Confocal microscopy and fluorescent probes were used to qualitatively and quantitatively analyze, at the subcellular level, living HN9.10e neurons during and after TlCl exposure. An early onset mitochondrial dysfunction appeared, associated with signs of cellular deregulation such as neurite shortening, loss of substrate adhesion, and increase of cytoplasmic calcium. The dose-dependent alteration of mitochondrial ROS (mtROS) level and of transmembrane mitochondrial potential (ΔΨm) has been observed also for very low TlCl doses (1 μg/L). The treatment with the ATP synthase inhibitor oligomycin revealed a severe impairment of the mitochondrial function, more significant than that measured by the simple quantification of the tetramethylrhodamine methyl ester (TMRM) fluorescence. These results highlight that mitochondria are a key subcellular target of TlCl neurotoxicity. The transmembrane mitochondrial potential was significantly correlated with the ethanol concentration in cell culture medium ( P < 0.001, r = -0.817), suggesting that ethanol could be potentially used as a biomarker of mitochondrial impairment.

Lactate and ethanol (EtOH) were determined in cell culture medium (CCM) of immortalized hippocampal neurons (HN9.10e cell line) before and after incubation with Thallium (Tl). This cell line is a reliable, in vitro model of one of the most vulnerable regions of central nervous system. Cells were incubated for 48 h with three different single Tl doses: 1, 10, 100 μg/L (corresponding to 4.9, 49 and 490 nM, respectively). After 48 h, neurons were "reperfused" with fresh CCM every 24/48 h until 7 days after the treatment and the removed CCM was collected and analysed. Confocal microscopy was employed to observe morphological changes. EtOH was determined by head space-solid phase microextraction -gas chromatography -mass spectrometry (HS-SPME-GCMS), lactate by RP-HPLC with UV detection. Tl exposure had significant effects on neuronal growth rate and morphology. The damage degree was dose-dependent. In not exposed cells, EtOH concentration was 0.18 ± 0.013 mM, which represents about 5% of lactate concentration (3.4 ± 0.10 mM). After Tl exposure lactate and EtOH increased. In CCM of 100 and 10 μg/L Tl-treated cells, lactate increased 24 h after reperfusion up to 2 and 3.3 times the control value, respectively. In CCM of 10 and 100 μg/L Tl-treated cells 24 h after reperfusion, EtOH increased up to 0.3 and 0.58 mmol/L. respectively. These results are consistent with significant alterations in energy metabolism, despite the low doses of Tl employed and the relatively short incubation time.

Lactate and ethanol (EtOH) were determined in cell culture medium (CCM) of immortalized hippocampal neurons (HN9.10e cell line) before and after incubation with Thallium (Tl). This cell line is a reliable, in vitro model of one of the most vulnerable regions of central nervous system. Cells were incubated for 48 h with three different single Tl doses: 1, 10, 100 μg/L (corresponding to 4.9, 49 and 490 nM, respectively). After 48 h, neurons were "reperfused" with fresh CCM every 24/48 h until 7 days after the treatment and the removed CCM was collected and analysed. Confocal microscopy was employed to observe morphological changes. EtOH was determined by head space-solid phase microextraction -gas chromatography -mass spectrometry (HS-SPME-GCMS), lactate by RP-HPLC with UV detection. Tl exposure had significant effects on neuronal growth rate and morphology. The damage degree was dose-dependent. In not exposed cells, EtOH concentration was 0.18 ± 0.013 mM, which represents about 5% of lactate concentration (3.4 ± 0.10 mM). After Tl exposure lactate and EtOH increased. In CCM of 100 and 10 μg/L Tl-treated cells, lactate increased 24 h after reperfusion up to 2 and 3.3 times the control value, respectively. In CCM of 10 and 100 μg/L Tl-treated cells 24 h after reperfusion, EtOH increased up to 0.3 and 0.58 mmol/L. respectively. These results are consistent with significant alterations in energy metabolism, despite the low doses of Tl employed and the relatively short incubation time.