Papers by Sandra Ceccatelli
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Toxicology Letters, 2006
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Toxicology and Applied Pharmacology, 2003
The 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is a highly toxic, widespread environmenta... more The 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) is a highly toxic, widespread environmental contaminant. Most of the toxic damage caused by TCDD is considered to be secondary to the binding of TCDD to the aryl hydrocarbon receptor, the AH receptor (AHR). TCDD is known to affect the vitamin A homeostasis. The effects of TCDD on body weight and on the expressions of AHR-associated genes may be modulated by the retinoid system. Therefore, we investigated the regulation pattern of expression of the AHR associated genes after TCDD exposure in wild-type 129/SV/C57BL/6 mice and in mice deficient in cellular retinol-binding protein type I (CRBP-I) of the same mixed genetic background. A single oral dose of TCDD (50 or 250 mug/kg) was administrated by gavage. Different brain areas, including cortex, hypothalamus, cerebellum, and olfactory bulb, as well as pituitary and liver, were dissected from CRBP-I knockout homozygous mice (-/-) and wild-type mice (+/+) killed at two time points (7 or 28 days after treatment). Compared with vehicle-treated controls, the relative levels of cytochrome P450 1A1 (CYP1A1) mRNA and protein expression in TCDD-treated animals were dramatically increased in mice of both CRBP-I genotypes (-/-, +/+). CYP1A1 mRNA levels increased up to 1400-fold in the pituitary, 110-fold in the brain, and up to 1600-fold in the liver. A general observation was that the relative induction of CYP1A1 and AHR transcription after TCDD dosing was highest in the +/+ mice. A high TCDD-induced aryl hydrocarbon receptor repressor (AHRR) mRNA expression was observed in the liver and in brain tissues. Interestingly, however, mice that lacked the CRBP-I protein (-/-) were found to have a significantly higher basal expression of AHRR gene in the pituitary compared to the wild-type (CRBP-I +/+) mice and in accordance a less pronounced induction of CYP1A1 and AHR was observed in the pituitary of the -/- mice. Immunohistochemical double staining analyses of the expression pattern of CYP1A1 and AHRR proteins in the pituitary revealed a colocalization of these two proteins. We conclude that the vitamin A homeostasis seems to have some influence to the TCDD-induced activation of AHR-regulated gene transcription in the brain and pituitary of the adult mouse.
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Toxicology Letters, 2003
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Toxicology, 2005
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Pathophysiology, 1998
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NeuroToxicology, 2002
The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related substances cause a wide variety of pat... more The 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related substances cause a wide variety of pathological alterations, with the most severe being progressive anorexia and body weight loss. These features suggest a possible involvement of the nervous system and endocrine organs, including the pituitary gland. TCDD-related toxicity is considered mainly to be mediated by the aryl hydrocarbon receptor (AHR) protein, which binds TCDD, and heterodimerizes with its partner protein, the aryl hydrocarbon receptor nuclear translocator (ARNT), and binds to xenobiotica responsive elements (XREs) in the promoter regions of biotransformation genes as well as genes involved in growth, differentiation and cellular homeostasis. In the present study, we have investigated the expression of AHR responsive genes in the pituitary of untreated and TCDD treated 129/SV/C57BL/6 mice in vivo and in pituitary cells in vitro. After TCDD or beta-naphthoflavone (beta NF) treatment, the relative levels of cytochrome P4501A1 (CYP1A1) mRNA and protein were dramatically increased in pituitary cells. The AHR repressor (AHRR) mRNA level was induced 7-13-fold by TCDD and beta NF. Furthermore, the expression of the adrenocorticotrophic hormone (ACTH) precursor, the proopiomelanocortin (POMC) gene, was investigated. A three-fold increase in POMC mRNA was observed in the pituitary of TCDD treated mice. POMC mRNA level was also increased in the pituitary cell line AtT-20 after TCDD treatment. The proteins encoded by POMC translational products, ACTH and beta-endorphin, were found with immunocytochemistry staining to be increased in AtT-20 cells after TCDD exposure. The presence of several XRE sequences in the promoter region and in the first intron of the human POMC gene suggest that the up-regulation of POMC expression in the pituitary may play a role in the endocrine alterations induced by TCDD. All together, the results point to the pituitary gland being a direct target for TCDD.
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Neuroendocrinology, 1996
Expression and estrogen regulation of the genes for nitric-oxide (NO)-synthesizing enzymes (NO sy... more Expression and estrogen regulation of the genes for nitric-oxide (NO)-synthesizing enzymes (NO synthase, NOS) were investigated by in situ hybridization. This study focused on regions of the hypothalamus that contain estrogen receptors and regulate specific neuroendocrine functions related to female sexual behavior and food intake, among others. Ovariectomized (OVX) rats were treated with vehicle or 3 micrograms/100 g estradiol benzoate (EB) for 7 days. Brains were sectioned and hybridized with antisense riboprobes for neuronal NOS, macrophage NOS and endothelial NOS. In the hypothalamus, mRNA was clearly detectable only for the neuronal NOS with the probes used. A strong hybridization signal was observed in the supraoptic paraventricular and ventromedial nuclei (SON, PVN and VMN, respectively). Quantitative analysis showed an increase in neuronal NOS mRNA in the VMN of the OVX rats treated with EB. The increase was mainly in the ventrolateral aspect of the VMN. No significant changes were observed in the hypothalamic SON and PVN. The data suggest that the expression of neuronal NOS mRNA in VMN can be regulated by estrogen.
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Journal of Neuroscience Research, 2000
Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methyl... more Cerebellar granule cells (CGC) have provided a reliable model for studying the toxicity of methylmercury (MeHg), a well‐known neurotoxicant contaminating the environment. In the present study we report that doses of MeHg ranging from 0.1 μM to 1.5 μM activated apoptosis, as shown by cell shrinkage, nuclear condensation, and formation of high‐molecular‐weight DNA fragments. Nevertheless, caspase‐3‐like activity was not significantly induced, and the broad caspase inhibitor Z‐VAD‐FMK was not capable of protecting the cells. This argues for a minor role of caspases in the intracellular pathways leading to MeHg‐induced cell death in CGC. Instead, proteolytic fragments obtained by specific calpain cleavage of procaspase‐3 and α‐fodrin were increased consistently in samples exposed to MeHg, pointing to a substantial activation of calpain. Notably, two antioxidants, 17β‐estradiol (10 μM) and the Δ8,9‐dehydro derivative of 17α‐estradiol J811 (10 μM), protected from MeHg damage, preventing morphological alterations, chromatin fragmentation, and activation of calpain. These findings underscore the key role of oxidative stress in MeHg toxicity, placing it upstream of calpain activation. The shielding effect of the 17β‐estradiol and the radical scavenger J811 is potentially relevant for the development of therapeutic strategies for MeHg intoxication. J. Neurosci. Res. 62:557–565, 2000. © 2000 Wiley‐Liss, Inc.
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Journal of Immunological Methods, 1999
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Free Radical Biology and Medicine, 2001
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Food and Chemical Toxicology, 2006
The study of interactions for those substances which tend to accumulate in food and affect the ne... more The study of interactions for those substances which tend to accumulate in food and affect the nervous system appears to be a fundamental point to characterize the combined exposure in vitro. In this study we included two food contaminants which are known neurotoxicants: methyl-mercury (Me-Hg) and the ortho-substituted PCB 153. PC12 cells were treated with Me-Hg (range 1e− 7, 2e− 6M) and PCB153 (range 1e− 5, 4e− 4M) in single and combined synchronous experiments and a mathematical model was set up ...
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European Journal of Neuroscience, 1999
Oxidative stress has been implicated in various neurodegenerative diseases. There is substantial ... more Oxidative stress has been implicated in various neurodegenerative diseases. There is substantial evidence indicating that gonadal hormones can affect neuronal cell survival via both a genomic as well as a non‐genomic mode of action. In the present study, the potential protective activity of testosterone on neuronal cells was investigated by using an in vitro/ex vivo model. Cerebellar granule cells (CGC) were prepared from 7‐day‐old rats which had been treated with a single dose of oil or testosterone propionate on postnatal day 3. After 7 days in culture, cells were exposed to oxidative challenges, including hydrogen peroxide and the nitric oxide donor S‐nitrosocysteine (SNOC), which can induce CGC death via apoptosis. Colchicine, which causes apoptosis via a different mechanism, was also used. The cells were monitored for apoptotic morphology by propidium iodide and TUNEL staining. Additionally, the presence of chromatin fragmentation was determined. CGC obtained from testosterone‐...
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European Journal of Neuroscience, 1999
The microtubule-disrupting agent colchicine is known to be neurotoxic toward certain neuronal pop... more The microtubule-disrupting agent colchicine is known to be neurotoxic toward certain neuronal populations including cerebellar granule cells (CGCs). In this study we investigated the involvement of cytochrome c release and caspase-3 activation during colchicine-induced CGC apoptosis. Treatment of rat CGCs with 1 micrometer colchicine (for up to 24 h) caused high molecular weight DNA fragmentation and nuclear condensation. An involvement of group II caspases (which includes caspase-3) was demonstrated by the proteolytic degradation of poly(ADP-ribose) polymerase (PARP) after 18 h exposure to colchicine. Colchicine induced a time-dependent increase in Ac-Asp-Glu-Val-Asp-alpha-(4-methyl-coumaryl-7-amide) (DEVD-MCA) cleavage activity in CGCs, which was blocked with a specific, peptide-based, aldehyde inhibitor of group II caspases, i. e. DEVD-CHO. We also observed a time-dependent proteolysis of caspase-3 as judged by the appearance of p17 which is one of the subunits of active caspase-3. Activation of caspase-3 during colchicine-induced apoptosis may be mediated by cytochrome c since there was a close correlation between the time courses of cytochrome c release from the mitochondria and of caspase-3 activation. Furthermore, colchicine-induced apoptosis, as assessed by propidium iodide visualization of the nuclei, could be blocked by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp (O-methyl) fluoromethyl ketone.
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Chemico-Biological Interactions, 2010
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Chemical Research in Toxicology, 2004
A solid phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS) method was deve... more A solid phase microextraction (SPME) gas chromatography/mass spectrometry (GC/MS) method was developed to assess actual doses of highly reactive organic compounds like styrene oxide (SO) in exposed cell cultures. Using SPME, we set up a method to measure accurately extracellular SO concentrations as well as to obtain an approximate assessment of intracellular levels. The SPME-GC/MS method was developed and validated using two different coating materials, carboxen-PDMS and polyacrylate. In cell-free systems, linearity was established over 3 orders of magnitude for both fibers, but carboxen-PDMS showed higher extraction efficiency and a lower limit of detection (0.5 x 10(-7) vs 10(-6) M for polyacrylate). Precision calculated as % RSD was within 4-16% for all intra- and interday determinations. Experiments performed to study SO stability in cell-free medium showed a time-dependent decrease in SO concentration (11% of initial the concentration after 24 h), mostly due to the spontaneous hydrolysis of SO into styrene glycol, which was measured by liquid chromatography/tandem mass spectrometry (LC/MS/MS). When the neuronal cell line (SK-N-MC) was exposed to a nominal concentration of 0.3 x 10(-4) M SO, the actual concentration measured in the supernatant was considerably lower and was found to decrease during incubation. Intracellular SO was estimated indirectly, by difference between the amount measured in the medium without cells and in the supernatant of the cell-containing medium.
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Brain Research, 2006
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Brain Research, 2001
It is known that steroid hormones can affect neuronal susceptibility to different types of insult... more It is known that steroid hormones can affect neuronal susceptibility to different types of insults, including oxidative stress. Using an in vitro/ex vivo model, we have previously shown that cerebellar granule cells prepared from neonatal rats treated with a single dose of testosterone are less vulnerable to oxidative stress-induced cell death, via a mechanism involving an upregulation of the cellular antioxidant defenses. Whether the testosterone protective action on cerebellar granule cells was direct or indirect remained to be clarified. Therefore, in this study we have investigated the effects of in vitro testosterone treatment, to see whether it also protects cerebellar granule cells from oxidative stress-induced damage. Cerebellar granule cells treated with 10(-6) M testosterone for 48 h were found less susceptible to damage induced by 50 microM hydrogen peroxide, as shown by a 30% decrease in the number of cells with apoptotic morphology. The addition of the androgen receptor antagonist flutamide abolished the protective effect of testosterone, suggesting an androgen receptor-mediated mechanism. This hypothesis was further supported by the presence of the androgen receptor in cultured cerebellar granule cells. The activity of the antioxidant enzyme catalase was also measured, and a 2-fold increase was detected in the testosterone treated cells, but not in the cells co-treated with flutamide. The present results demonstrate that cerebellar granule cells treated in vitro with testosterone are protected from oxidative stress via a mechanism mediated by the androgen receptor. Similarly to what we observed after in vivo administration of testosterone, the potentiation of the antioxidant defences seems to play a major role in the protection afforded by testosterone.
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Apoptosis, 2008
Oxidative stress occurs as a consequence of disturbance in the balance between the generation of ... more Oxidative stress occurs as a consequence of disturbance in the balance between the generation of reactive oxygen species (ROS) and the antioxidant defence mechanisms. The interaction of ROS with DNA can cause single-, or double-strand breaks that subsequently can lead to the activation of p53, which is central for the regulation of cellular response, e.g. apoptosis, to a range of environmental and intracellular stresses. Previous reports have suggested a regulatory role of p53 in the early activation of caspase-2, upstream of mitochondrial apoptotic signaling. Here we show that excessive ROS formation, induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) exposure, induces apoptosis in primary cultured neural stem cells (NSCs) from cortices of E15 rat embryos. Following DMNQ exposure cells exhibited apoptotic hallmarks such as Bax oligomerization and activation, cytochrome c release, caspase activation and chromatin condensation. Additionally, we could show early p53 accumulation and a subsequent activation of caspase-2. The attenuation of caspase-2 activity with selective inhibitors could antagonize the mitochondrial signaling pathway and cell death. Overall, our results strongly suggest that DMNQ-induced oxidative stress causes p53 accumulation and consequently caspase-2 activation, which in turn initiates apoptotic cell death via the mitochondria-mediated caspase-dependent pathway in NSCs.
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Papers by Sandra Ceccatelli