Elisa Roda

Human developmental neurotoxicity of methylmercury : Impact of variables and risk modifiers

Methylmercury (MeHg) is a widespread environmental and food toxicant which has long been known to affect neurodevelopment in both humans and experimental animals. Risk assessment for MeHg is mainly based on human data coming from the massive episodes of poisoning in Japan and Iraq, as well as from large scale epidemiological studies concerning childhood development and neurotoxicity in relation to in utero exposure in various fish eating communities around the world. Despite the extensive literature and research, the threshold dose for MeHg neurotoxic effects is still unclear, in particular when it comes to subtle effects on neurobehaviour. In this article clinical and epidemiological findings concerning the neurodevelopmental toxicity of MeHg are reviewed. Much attention is focussed on the potential impact of factors, such as diet and nutrition, gender, pattern of exposure and co-exposure to other neurotoxic pollutants, which may modulate MeHg toxic effects. These factors, together with the notion that some symptoms may ensue or exacerbate with aging, contribute to the difficulties in the definition of safe levels for developmental exposure.

Effects of water-soluble functionalized multi-walled carbon nanotubes examined by different cytotoxicity methods in human astrocyte D384 and lung A549 cells

The widespread projected use of functionalized carbon nanotubes (CNTs) makes it important to understand their potential harmful effects. Two cell culture systems, human A549 pneumocytes and D384 astrocytoma cells, were used to assess cytotoxicity of multi-walled CNTs (MWCNTs) with varying degrees of functionalization. Laboratory-made highly functionalized hf-MW-NH(2) and less functionalized CNTs (MW-COOH and MW-NH(2)) were tested in comparison with pristine MWCNTs, carbon black (CB) and silica (SiO(2)) by MTT assay and calcein/propidium iodide (PI) staining. Purity and physicochemical properties of the test nanomaterials were also determined. In both MTT and calcein/PI assays, highly functionalized CNTs (hf-MW-NH(2)) caused moderate loss of cell viability at doses >or=100 microg/ml being apparently less cytotoxic than SiO(2). In preparations treated with CB or the other nanotube types (pristine MWCNTs, MW-COOH and the less functionalized amino-substituted MW-NH(2)) the calcein/PI test indicated no loss of cell viability, whereas MTT assay apparently showed apparent cytotoxic response, occurring not dose-dependently at exceedingly low CNT concentrations (1 microg/ml). The latter nanomaterials were difficult to disperse showing higher aggregate ranges and tendency to agglomerate in bundle-like form in cell cultures. In contrast, hf-MW-NH(2) were water soluble and easily dispersible in medium; they presented lower aggregate size range as well as considerably lower length to diameter ratios and low tendency to form aggregates compared to the other CNTs tested. The MTT data may reflect a false positive cytotoxicity signal possibly due to non-specific CNT interaction with cell culture components. Thus, these properties obtained by chemical functionalization, such as water solubility, high dispersibility and low agglomeration tendency were relevant factors in modulating cytotoxicity. This study indicates that properties obtained by chemical functionalization, such as water solubility, high dispersibility and low agglomeration tendency are relevant factors in modulating cytotoxicity of CNTs.

Neurodevelopmental toxicity of methylmercury: Laboratory animal data and their contribution to human risk assessment

Methylmercury (MeHg) is one of the most significant public health hazards. The clinical findings in the victims of the Japanese and Iraqi outbreaks have disclosed the pronounced susceptibility of the developing brain to MeHg poisoning. This notion has triggered worldwide scientific attention toward the long-term consequences of prenatal exposure on child development in communities with chronic low level dietary exposure. MeHg neurodevelopmental effects have been extensively investigated in laboratory animals under well-controlled exposure conditions. This article provides an updated overview of the main neuromorphological and neurobehavioral changes reported in non-human primates and rodents following developmental exposure to MeHg. Different aspects of MeHgs effects on the immature organism are reported, with particular reference to the delayed onset of symptoms and the persistency of central nervous system (CNS) injury/dysfunction. Particular attention is paid to the comparative toxicity assessment across species, and to the degree of concordance/discordance between human and animal data. The contribution of animal studies to define the role of potential effect modifiers and variables on MeHg dose-response relationships is also addressed. The ultimate goal is to discuss the relevance of laboratory animal results, as a complementary tool to human data, with regard to the human risk assessment process.

Pulmonary toxicity of instilled cadmium-doped silica nanoparticles during acute and subacute stages in rats.

Potential risk associated with new nanomaterial exposure needs to be assessed. This in vivo study investigated pulmonary effects of engineered cadmium-containing silica nanoparticles Cd/SiNPs (1 mg/rat), silica SiNPs (600 μg/rat) and CdCl₂ (400 μg/rat) 1, 7 and 30 days after intratracheal instillation. Comprehensive histopathological and immunocytochemical characterization of lung damage in terms of apoptosis, cell proliferation, inflammation, fibrosis and metabolism were obtained. After exposure to all treatments, lung parenchyma showed injury patterns characterized by collapsed alveoli, inflammation, granuloma formation, thickened alveolar septa and bronchiolar epithelium exfoliation. Type II pneumocytes, containing scarcely surfactant-lamellated bodies, were also observed. Apoptotic phenomena enhanced as following, Cd/SiNPs>CdCl₂> SiNPs. In parallel with these findings, a significant increase of PCNA-immunoreactive cells was detected together with high mitotic activity. Cellular localization and distribution of IL-6, IP-10 and TGF-β1 revealed an increased expression of these cytokines as evidence of an enhanced cellular inflammatory response. CYP450-immunoreactivity was also enhanced, at bronchiolar (e.g. Clara cells) and alveolar (e.g. macrophages) level after both Cd/SiNPs and CdCl₂. These overall effects were observed acutely and lasted until the 30th day, with Cd/SiNPs producing the most marked effects. Collagen-immunolabelling changed particularly 7 and 30 days after Cd/SiNPs, when a strong stromal fibrogenic reaction occurred. The present findings suggest that Cd/SiNPs produce significantly greater pulmonary alterations than either SiNPs or CdCl₂ under the present experimental conditions.

Developmental plasticity of rat cerebellar cortex after cisplatin injury: Inhibitory synapses and differentiating Purkinje neurons

A single injection of cisplatin, a cytostatic agent, (5 microg/g body weight) in 10-day old rats leads later to the reorganization of the cerebellar cortex in lobules VI-VIII of the vermis. Double immunofluorescence reaction for glutamate receptor (GluR)2/3, a ionotropic glutamate receptor that labels postsynaptically Purkinje neurons, and glutamic acid decarboxylase (GAD)65, an isoform of the GABA synthesis enzyme that labels presynaptically inhibitory terminals in the molecular layer, were employed. Less-differentiated Purkinje cells were present in rats treated on postnatal day (PD)11 at the top of lobule VI and in lobules VII-VIII, in comparison with the deep zones of the same lobules and lobule III. The changes were interpreted as due to loss of trophic factors of Purkinje cell growth, e.g. signaling molecules and granule cells. However, we have shown that a remodelling of Purkinje cell dendrites occurred on PD30 (20 days after cisplatin). In fact, despite of the GluR2/3 labeling of the entire Purkinje cell dendrites, the GAD65 immunofluorescent terminals were adjacent to the proximal parts of the dendrite, while they were scarce in the distal dendritic branchlets. The findings were discussed in relation to the changed cytoarchitecture of the cerebellar cortex, which from PD17 to PD30 includes regeneration of the external germinal layer, reorientation of the main dendritic branches and of the Purkinje cell branchlets, and the presence of ectopic cells.

Safety Evaluation of Engineered Nanomaterials for Health Risk Assessment: An Experimental Tiered Testing Approach Using Pristine and Functionalized Carbon Nanotubes

Increasing application of engineered nanomaterials within occupational, environmental, and consumer settings has raised the levels of public concern regarding possible adverse effects on human health. We applied a tiered testing strategy including (i) a first in vitro stage to investigate general toxicity endpoints, followed by (ii) a focused in vivo experiment. Cytotoxicity of laboratory-made functionalized multiwalled carbon nanotubes (CNTs) (i.e., MW-COOH and MW-NH2), compared to pristine MWCNTs, carbon black, and silica, has been assessed in human A549 pneumocytes by MTT assay and calcein/propidium iodide (PI) staining. Purity and physicochemical properties of the test nanomaterials were also determined. Subsequently, pulmonary toxic effects were assessed in rats, 16 days after MWCNTs i.t. administration (1 mg/kg b.w.), investigating lung histopathology and monitoring several markers of lung toxicity, inflammation, and fibrosis. In vitro data: calcein/PI test indicated no cell viability loss after all CNTs treatment; MTT assay showed false positive cytotoxic response, occurring not dose dependently at exceedingly low CNT concentrations (1 μg/mL). In vivo results demonstrated a general pulmonary toxicity coupled with inflammatory response, without overt signs of fibrosis and granuloma formation, irrespective of nanotube functionalization. This multitiered approach contributed to clarifying the CNT toxicity mechanisms improving the overall understanding of the possible adverse outcomes resulting from CNT exposure.

Single step determination of PCB 126 and 153 in rat tissues by using solid phase microextraction/gas chromatography-mass spectrometry : Comparison with solid phase extraction and liquid/liquid extraction

A simple and reliable solid phase microextraction/gas chromatography-mass spectrometry (SPME/GC-MS) method was developed for the single-step determination of PCBs 126 and 153 in rat brain and serum, using liquid/liquid and solid phase extraction (SPE) as reference techniques. The multi-factor categorical experimental design used to study simultaneously the main parameters and their interactions affecting the efficiency of the method, showed that the use of an 85mum PA exposed at 100 degrees C for 40min was the optimum sampling condition for both PCBs. SPME was then validated by studying its linear dynamic (over two orders of magnitude), limits of detection (brain: 2ng/g, serum: 0.2ng/g) and analytical precision that was within 9% for SPME in both brain and serum. Finally, the method was used to determine the brain and blood target dose in mothers and pups after oral exposure of the mothers.

Cerebellum cholinergic muscarinic receptor (subtype-2 and -3) and cytoarchitecture after developmental exposure to methylmercury: An immunohistochemical study in rat

The developing central nervous system (CNS) is a target of the environmental toxicant methylmercury (MeHg), and the cerebellum seems the most susceptible tissue in response to this neurotoxicant. The cholinergic system is essential for brain development, acting as a modulator of neuronal proliferation, migration and differentiation processes; its muscarinic receptors (MRs) play pivotal roles in regulating important basic physiologic functions. By immunohistochemistry, we investigated the effects of perinatal (GD7-PD21) MeHg (0.5 mg/kg bw/day in drinking water) administration on cerebellum of mature (PD36) and immature (PD21) rats, evaluating the: (i) M2- and M3-MR expression; (ii) presence of gliosis; (iii) cytoarchitecture alterations. Regarding to M2-MRs, we showed that: at PD21, MeHg-treated animals did not display any differences compared to controls, while, at PD36 there was a significant increase of M2-immunopositive Bergmann cells in the molecular layer (ML), suggesting a MeHg-related cytotoxic effect. Similarly to M2-MRs, at PD21 the M3-MRs were not affected by MeHg, while, at PD36 a lacking immunoreactivity of the granular layer (IGL) was observed after MeHg treatment. In MeHg-treated rats, at both developmental points, we showed reactive gliosis, e.g. a significant increase in Bergmann glia of the ML and astrocytes of the IGL, identified by their expression of glial fibrillar acidic protein. No MeHg-related effects on Purkinje cells were detected neither at weaning nor at puberty. These findings suggest: (i) a delayed MeHg exposure-related effect on M2- and M3-MRs, (ii) an overt MeHg-related cytotoxic effect on cerebellar oligodendroglia, e.g. reactive gliosis, (iii) a selective vulnerability of granule cells and Purkinje neurons to MeHg, with the latter that remain unharmed.

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.

Apoptosis induction and histological changes in rat kidney following Cd-doped silica nanoparticle exposure: evidence of persisting effects

Abstract Histological and immunocytochemical methods were used to examine rat’s renal responses to intratracheal (i.t.) instillation of model cadmium-containing silica nanoparticles (Cd-SiNPs) and also exploring whether these potential modifications would be associated with toxicogenomic changes. Renal effects of Cd-SiNPs (1 mg/rat), CdCl2 (400 µg/rat), SiNPs (600 µg/rat) or 0.1 ml saline (control), assessed 7 and 30 d post-i.t., included (i) induction of apoptosis, (ii) cell proliferation and (iii) the overall toxic response evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining, proliferating cell nuclear antigen (PCNA) immunohistochemistry as well as Periodic acid Schiff and Hematoxylin & Eosin, respectively. Area-specific apoptosis was observed in all treatment groups, the cortex and inner medulla being the most affected regions: the apoptotic changes were apparent seven days post-exposure in both areas and were still observable in inner medulla at day 30. Apoptotic frequency increase was more pronounced in Cd-SiNP-treated animals compared to either CdCl2 or SiNPs groups. At day 7, the observed parallel increased number of PCNA immunopositive cells may be associated with an enhanced cell proliferation aimed at replacing the damaged cells. Histopathological findings demonstrated comparable morphological changes of the renal structure (at glomerular and tubular levels) occurring after all treatments at both time-points and more markedly 30 d after instillation. Both morphological and toxicogenomic evaluations confirmed long-lasting renal effects of Cd-SiNPs on apoptosis and regulatory processes. Bare SiNPs i.t. administration caused morphological and apoptotic changes but did not modify gene expression profile in kidney. These findings substantiate the notion that multiple assays and an integrated testing strategy should be recommended to characterize toxicological responses to nanoparticles in mammalian systems.