Toxicology Unit

Cáceres, Spain

Toxicology Unit

Cáceres, Spain
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Masi A.,University of Florence | Masi A.,Toxicology Unit | Narducci R.,University of Florence | Resta F.,University of Florence | And 4 more authors.
European Journal of Neuroscience | Year: 2015

The selective vulnerability of substantia nigra pars compacta (SNc) dopaminergic (DA) neurons is an enigmatic trait of Parkinson's disease (PD), especially if compared to the remarkable resistance of closely related DA neurons in the neighboring ventral tegmental area (VTA). Overall evidence indicates that specific electrophysiological, metabolic and molecular factors underlie SNc vulnerability, although many pieces of the puzzle are still missing. In this respect, we recently demonstrated that 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of the parkinsonizing toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), alters the electrophysiological properties of SNc DA neurons in vitro by inhibiting the hyperpolarization-activated current (Ih). Here, we present an electrophysiological investigation of the functional role of Ih in the integration of synaptic inputs in identified SNc and VTA DA neurons, comparatively, in acute midbrain slices from TH-GFP mice. We show that pharmacological suppression of Ih increases the amplitude and decay time of excitatory postsynaptic potentials, leading to temporal summation of multiple excitatory potentials at somatic level. Importantly, these effects are quantitatively more evident in SNc DA neurons. We conclude that Ih regulates the responsiveness to excitatory synaptic transmission in SNc and VTA DA neurons differentially. Finally, we present the hypothesis that Ih loss of function may be linked to PD trigger mechanisms, such as mitochondrial failure and ATP depletion, and act in concert with SNc-specific synaptic connectivity to promote selective vulnerability. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Viticchie G.,University of Rome Tor Vergata | Agostini M.,University of Rome Tor Vergata | Agostini M.,Toxicology Unit | Lena A.M.,University of Rome Tor Vergata | And 10 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Short p63 isoform, ΔNp63, is crucial for epidermis formation, and it plays a pivotal role in controlling the turnover of basal keratinocytes by regulating the expression of a subset of genes involved in cell cycle and cell adhesion programs. The glycolytic enzyme hexokinase 2 (HK2) represents the first step of glucose utilization in cells. The family of HKs has four isoforms that differ mainly in their tissue and subcellular distribution. The preferential mitochondrial localization of HK2 at voltage-dependent anion channels provides access to ATP generated by oxidative phosphorylation and generates an ADP/ATP recycling mechanism to maintain high respiration rates and low electron leak. Here, we report that ΔNp63 depletion in human keratinocytes impairs mitochondrial basal respiration and increases mitochondrial membrane polarization and intracellular reactive oxygen species. We show ΔNp63- dependent regulation of HK2 expression, and we use ChIP, validated by p63-Chip sequencing genomewide profiling analysis, and luciferase assays to demonstrate the presence of one p63-specific responsive element within the 15th intronic region of the HK2 gene, providing evidence of a direct interaction. Our data support the notion of ΔNp63 as a master regulator in epithelial cells of a combined subset of molecular mechanisms, including cellular energy metabolism and respiration. The ΔNp63-HK2 axis is also present in epithelial cancer cells, suggesting that ΔNp63 could participate in cancer metabolic reprogramming.

Resta F.,University of Florence | Masi A.,University of Florence | Masi A.,Toxicology Unit | Sili M.,University of Florence | And 4 more authors.
Neuropharmacology | Year: 2016

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels have a key role in the control of cellular excitability. HCN2, a subgroup of the HCN family channels, are heavily expressed in small dorsal root ganglia (DRG) neurons and their activation seems to be important in the determination of pain intensity. Intracellular elevation of cAMP levels activates HCN-mediated current (Ih) and small DRG neurons excitability. GPR35, a Gi/o coupled receptor, is highly expressed in small DRG neurons, and we hypothesized that its activation, mediated by endogenous or exogenous ligands, could lead to pain control trough a reduction of Ih current. Patch clamp recordings were carried out in primary cultures of rat DRG neurons and the effects of GPR35 activation on Ih current and neuronal excitability were studied in control conditions and after adenylate cyclase activation with either forskolin or prostaglandin E2 (PGE2). We found that both kynurenic acid (KYNA) and zaprinast, the endogenous and synthetic GPR35 agonist respectively, were able to antagonize the forskolin-induced depolarization of resting membrane potential by reducing Ih-mediated depolarization. Similar results were obtained when PGE2 was used to activate adenylate cyclase and to increase Ih current and the overall neuronal excitability. Finally, we tested the analgesic effect of both GPR35 agonists in an in vivo model of PGE2-induced thermal hyperalgesia. In accord with the hypothesis, both KYNA and zaprinast showed a dose dependent analgesic effect. In conclusion, GPR35 activation leads to a reduced excitability of small DRG neurons in vitro and causes a dose-dependent analgesia in vivo. GPR35 agonists, by reducing adenylate cyclase activity and inhibiting Ih in DRG neurons may represent a promising new group of analgesic drugs. © 2016 Elsevier Ltd. All rights reserved.

Glinn M.,Toxicology Unit | Adatsi F.,Toxicology Unit | Curtis P.,Alcohol Enforcement Unit
Journal of Forensic Sciences | Year: 2011

The State of Michigan uses the Datamaster as an evidential breath testing device. The newest version, the DMT, will replace current instruments in the field as they are retired from service. The Michigan State Police conducted comparison studies to test the analytical properties of the new instrument and to evaluate its response to conditions commonly cited in court defenses. The effects of mouth alcohol, objects in the mouth, and radiofrequency interference on paired samples from drinking subjects were assessed on the DMT. The effects of sample duration and chemical interferents were assessed on both instruments, using drinking subjects and wet-bath simulators, respectively. Our testing shows that Datamaster and DMT results are essentially identical; the DMT gave accurate readings as compared with measurements made using simulators containing standard ethanol solutions and that the DMT did not give falsely elevated breath alcohol results from any of the influences tested. © 2011 American Academy of Forensic Sciences.

Gerhardt E.,University of Gottingen | Graber S.,Leibniz Institute for Molecular Pharmacology | Szego E.M.,University of Gottingen | Moisoi N.,Toxicology Unit | And 3 more authors.
PLoS ONE | Year: 2011

Heterozygous loss-of-function mutation of the human gene for the mitochondrial protease HtrA2 has been associated with increased risk to develop mitochondrial dysfunction, a process known to contribute to neurodegenerative disorders such as Huntington's disease (HD) and Parkinson's disease (PD). Knockout of HtrA2 in mice also leads to mitochondrial dysfunction and to phenotypes that resemble those found in neurodegenerative disorders and, ultimately, lead to death of animals around postnatal day 30. Here, we show that Idebenone, a synthetic antioxidant of the coenzyme Q family, and Resveratrol, a bioactive compound extracted from grapes, are both able to ameliorate this phenotype. Feeding HtrA2 knockout mice with either compound extends lifespan and delays worsening of the motor phenotype. Experiments conducted in cell culture and on brain tissue of mice revealed that each compound has a different mechanism of action. While Idebenone acts by downregulating the integrated stress response, Resveratrol acts by attenuating apoptosis at the level of Bax. These activities can account for the delay in neuronal degeneration in the striata of these mice and illustrate the potential of these compounds as effective therapeutic approaches against neurodegenerative disorders such as HD or PD. © 2011 Gerhardt et al.

Wilhelm M.T.,Princess Margaret Hospital | Wilhelm M.T.,Karolinska Institutet | Rufini A.,Princess Margaret Hospital | Rufini A.,Toxicology Unit | And 16 more authors.
Genes and Development | Year: 2010

Mice with a complete deficiency of p73 have severe neurological and immunological defects due to the absence of all TAp73 and ΔNp73 isoforms. As part of our ongoing program to distinguish the biological functions of these isoforms, we generated mice that are selectively deficient for the ΔNp73 isoform. Mice lacking ΔNp73 (ΔNp73-/- mice) are viable and fertile but display signs of neurodegeneration. Cells from ΔNp73 -/- mice are sensitized to DNA-damaging agents and show an increase in p53-dependent apoptosis. When analyzing the DNA damage response (DDR) in ΔNp73-/- cells, we discovered a completely new role for ΔNp73 in inhibiting the molecular signal emanating from a DNA break to the DDR pathway. We found that ΔNp73 localizes directly to the site of DNA damage, can interact with the DNA damage sensor protein 53BP1, and inhibits ATM activation and subsequent p53 phosphorylation. This novel finding may explain why human tumors with high levels of ΔNp73 expression show enhanced resistance to chemotherapy. © 2010 by Cold Spring Harbor Laboratory Press.

Couchman L.,Toxicology Unit | Birch M.,Toxicology Unit | Ireland R.,King's College | Corrigan A.,St Thomas Hospital | And 4 more authors.
Analytical and Bioanalytical Chemistry | Year: 2012

Tyrosine kinase inhibitors (TKIs) are used to treat a number of cancers, including chronic myeloid leukaemia and hepatocellular carcinoma. Therapeutic drug monitoring (TDM) may be indicated to (1) monitor adherence, (2) guide dosage, and (3) minimise the risk of drug-drug interactions and dose-related toxicity. On-line, automated sample preparation provided by TurboFlow technology (ThermoFisher Scientific) in conjunction with the sensitivity and selectivity of tandem mass spectrometry (MS/MS) detection may be applied to the analysis of single drugs and metabolites. We report the use of TurboFlow LC-MS/MS for the analysis of nine TKIs and metabolites (imatinib, N-desmethylimatinib, dasatinib, nilotinib, erlotinib, gefitinib, lapatinib, sorafenib, sunitinib) in human plasma or serum for TDM purposes. An Aria Transcend TLX-II system coupled with a TSQ Vantage was used. Samples (50 μL) were vortex mixed with internal standard solution (150 μL imatinib-D8, gefitinib-D8, sunitinib-D 10, and nilotinib- 13C 2 15N2 in acetonitrile) and, after centrifugation 100 μL supernatant were injected directly onto a 50×0.5-mm Cyclone TurboFlow column. Analytes were focussed onto a 50×2.1-mm (3 μm) Hypersil GOLD analytical column and eluted with an acetonitrile/ water gradient. Analytes were monitored in selected reaction monitoring mode (positive APCI). Total analysis time was 7 min without multiplexing. Calibration was linear (R2>0.99) for all analytes. Inter- and intra-Assay precision (in percent relative standard deviation, RSD) was <11 % and accuracy 89-117 % for all analytes. No matrix effects were observed. This method is suitable for high-Throughput TDM in patients undergoing chronic therapy with TKIs and has been utilised in the analysis of clinical samples. © Springer-Verlag 2012.

Kong Y.W.,Toxicology Unit | Ferland-McCollough D.,Toxicology Unit | Jackson T.J.,Toxicology Unit | Jackson T.J.,University of Nottingham | Bushell M.,Toxicology Unit
The Lancet Oncology | Year: 2012

Since the identification of microRNAs (miRNAs) in 1993, and the subsequent discovery of their highly conserved nature in 2000, the amount of research into their function-particularly how they contribute to malignancy-has greatly increased. This class of small RNA molecules control gene expression and provide a previously unknown control mechanism for protein synthesis. As such, it is unsurprising that miRNAs are now known to play an essential part in malignancy, functioning as tumour suppressors and oncogenes. This Review summarises the present understanding of how miRNAs operate at the molecular level; how their dysregulation is a crucial part of tumour formation, maintenance, and metastasis; how they can be used as biomarkers for disease type and grade; and how miRNA-based treatments could be used for diverse types of malignancies. © 2012 Elsevier Ltd.

Villena V.P.,Toxicology Unit
Journal of Analytical Toxicology | Year: 2010

Creatinine concentration is commonly used to verify the authenticity of urine specimens submitted for illicit drug screening. This study evaluated creatinine screening of donor urine specimens as a tool for detecting substituted and/or tampered specimens. The study carried out creatinine assay of animal urine, fruit juices, and urine from creatine-supplemented subjects by a modified version of the Jaffe reaction. All specimens were analyzed for creatinine concentration in a chemistry-immuno analyzer. Results showed that urine specimens from common domestic pets, including cats, dogs, and horses, have creatinine values similar to normal human values. Most fruit juices tested contained no detectable creatinine, and the few that did showed poor "urine" chemical integrity. Creatine supplementation by donors was found not to provide an effective means of elevating creatinine concentration in urine when attempting to flush out water-soluble drugs in the body. Thus, the assay for creatinine proved useful for the detection of some but not all adulterated urine specimens.

Ensibi C.,University of Carthage | Perez-Lopez M.,Toxicology Unit | Soler Rodriguez F.,Toxicology Unit | Miguez-Santiyan M.P.,Toxicology Unit | And 2 more authors.
Environmental Toxicology and Pharmacology | Year: 2013

The present study is aimed to evaluate the effect of sublethal exposure to the pyrethroid pesticide deltamethrin, according to biometric parameters and important liver biomarkers in the common carp (Cyprinus carpio). Fish were exposed for 4, 15 and 30 days to deltamethrin mixed to the aquaria water at the concentrations of 0.08, 0.4 and 0.8. μg/L. The exposure did not affect fish biometric parameters. Glutathione-S-transferase (GST) activity in liver (hepatopancreas) increased in fish exposed to the high concentration of pesticide only after 30 days when compared to controls. A significant increase of malondialdehyde (MDA) content (79.4% and 90.2% of control) was remarked after 15 and 30 days of exposure to the highest concentration of pesticide. Catalase activities (CAT) went up after 4 days of was to the concentration of 0.4. μg/L and 0.8. μg/L and at 15 days of exposure to 0.4. μg/L. The most significant change of glutathione reductase (GR) activity was recorded at the fourth day, when GR activity was significantly increased after exposure to the concentrations of 0.4. μg/L and 0.8. μg/L of deltamethrin. © 2013 Elsevier B.V.

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