Genotoxicity evaluation of three classes of manufactured nanomaterials on primary human lymphocytes

Human exposure to manufactured nanomaterials (MNMs) through consumers’ goods has increased worldwide, including titanium dioxide (TiO2), multiwalled carbon-nanotubes (MWCNT) and silicon dioxides (SiO2). Specific properties of MNMs, such as size and high surface area/mass, render them attractive for many applications, but may also be associated to higher toxicity in biological systems and adverse effects in humans. In the context of an EU Joint Action (NANOGENOTOX), aimed at establishing a robust methodology for evaluation of the MNMs genotoxicity, the objective of the present work was to characterize the potential genotoxic effects of a panel of well-characterized MNMs in human lymphocytes. Cultures of lymphocytes from healthy volunteers were exposed to several non-cytotoxic concentrations of four TiO2, six MWCNT and four SiO2 MNMs, dispersed according to a previously established procedure, for 30h; concurrent positive controls included a chemical (mitomycin C) and a reference nanosized ZnO. The cytokinesis-block micronucleus assay was performed according to the OECD guideline 487 and the frequencies of micronucleated binucleate (MNBNC) and mononucleate cells were determined. The results showed that slight but significant increases in the frequencies of MNBNC were observed for some TiO2 NMs and MWCNTs, as compared to controls, while all SiO2 NMs were negative in the micronucleus assay; a single dose of ZnO was able to significantly induce MNBNC. However, no clear monotonic dose-response relationships could be disclosed, not enabling clear conclusions about the genotoxicity of the tested MNMs. No influence on cell cycle progression and cell viability was noted by the cytokinesis-block proliferation index. In summary, although the micronucleus assay in primary human lymphocytes is suitable to evaluate the genotoxicity of MNMs following standardized dispersion and assay procedures, data from other cell lines or from other endpoints (e.g. comet assay) have to be gathered in order to achieve a robust genotoxicity evaluation

Conclusions of the French Food Safety Agency on the toxicity of bisphenol A

Since more than 10 years, risk assessment of bisphenol A (BPA) is debated at the international level. In 2008, the U.S. National Toxicology Program (NTP) expressed some concern for adverse effects, at current level of exposure to BPA, on developmental toxicity. In this context, the French Food Safety Agency (AFSSA) decided to review the toxicity data on BPA with a special focus on this endpoint at doses below 5mg/kg bw/day (the no observed adverse effect level set by different regulatory bodies). This paper summarizes the conclusions of a collective assessment conducted by an expert Working Group from AFSSA. Studies were classified into 3 groups: (i) finding no toxicity, (ii) reporting results not considered to be of concern and (iii) indicating warning signals. The term "warning signal" means that no formal conclusion can be drawn regarding the establishment of a health based guidance value but the study raises some questions about the toxicity of BPA at low doses. It was concluded that studies are needed to ascertain the significance for human health of these warning signals and to be able to propose new methodologies for assessing the risks associated with low doses of BPA and more generally of endocrine disruptors

Intrinsic antibacterial activity of nanoparticles made of β-cyclodextrins potentiates their effect as drug nanocarriers against tuberculosis

Multi-drug-resistant tuberculosis (TB) is a major public health problem, concerning about half a million cases each year. Patients hardly adhere to the current strict treatment consisting of more than 10 000 tablets over a 2-year period. There is a clear need for efficient and better formulated medications. We have previously shown that nanoparticles made of cross-linked poly-β-cyclodextrins (pβCD) are efficient vehicles for pulmonary delivery of powerful combinations of anti-TB drugs. Here, we report that in addition to being efficient drug carriers, pβCD nanoparticles are endowed with intrinsic antibacterial properties. Empty pβCD nanoparticles are able to impair Mycobacterium tuberculosis (Mtb) establishment after pulmonary administration in mice. pβCD hamper colonization of macrophages by Mtb by interfering with lipid rafts, without inducing toxicity. Moreover, pβCD provoke macrophage apoptosis, leading to depletion of infected cells, thus creating a lung microenvironment detrimental to Mtb persistence. Taken together, our results suggest that pβCD nanoparticles loaded or not with antibiotics have an antibacterial action on their own and could be used as a carrier in drug regimen formulations effective against TB.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Interlaboratory comparison study of the Colony Forming Efficiency assay for assessing cytotoxicity of nanomaterials

Nanotechnology has gained importance in the past years as it provides opportunities for industrial growth and innovation. However, the increasing use of manufactured nanomaterials (NMs) in a number of commercial applications and consumer products raises also safety concerns and questions regarding potential unintended risks to humans and the environment. Since several years the European Commission’s Joint Research Centre (JRC) is putting effort in the development, optimisation and harmonisation of in vitro test methods suitable for screening and hazard assessment of NMs. Work is done in collaboration with international partners, in particular the Organisation for Economic Co-operation and Development (OECD). This report presents the results from an interlaboratory comparison study of the in vitro Colony Forming Efficiency (CFE) cytotoxicity assay performed in the frame of OECD's Working Party of Manufactured Nanomaterials (WPMN). Twelve laboratories from European Commission, France, Italy, Japan, Poland, Republic of Korea, South Africa and Switzerland participated in the study coordinated by JRC. The results show that the CFE assay is a suitable and robust in vitro method to assess cytotoxicity of NMs. The assay protocol is well defined and is easily and reliably transferable to other laboratories. The results obtained show good intra and interlaboratory reproducibility of the assay for both the positive control and the tested nanomaterials. In conclusion the CFE assay can be recommended as a building block of an in vitro testing battery for NMs toxicity assessment. It could be used as a first choice method to define dose-effect relationships for other in vitro assays.JRC.I.4-Nanobioscience

Etude de la spécificité du test des comètes in vivo (application à l'étude de produits à tropisme rénal)

En France, le cancer du rein représente 3 % de l'ensemble des cancers et se situe par sa fréquence au 7ème rang chez l'homme et au 9ème chez la femme. Au cours des deux dernières décennies, l'incidence des cancers du rein chez l'Homme a augmenté aussi bien aux Etats-Unis, dans les autres pays occidentaux et au Japon qu'à travers le monde. De part leurs fonctions de filtration, réabsorption et sécrétion, les reins sont effectivement exposés à des concentrations élevées de substances potentiellement néphrotoxiques qui peuvent être présentes dans l'environnement, dans le milieu professionnel ou utilisées en thérapie. A ce jour, l'évaluation de la génotoxicité d'un produit fait appel à une batterie de tests dont certains sont bien reconnus mais la recherche de nouvelles techniques capables d'identifier le(s) organe(s) cible(s) et/ou de refléter les divers mécanismes d'action mis en jeu lors d'une atteinte du matériel génétique, reste toujours d'actualité. Dans ce contexte, le test des comètes in vivo, méthode très sensible d'évaluation de la génotoxicité applicable à tout type cellulaire ou à tout tissu qui permet de quantifier, au niveau de cellules individualisées, les cassures de brins de l'ADN ainsi que les sites alcali-labiles induits par différents traitements, a été mis en oeuvre en conditions alcalines (pH>13) sur cellules isolées de rein de rat. Au cours de cette étude, nous avons tout d'abord montré que le test des comètes in vivo sur cellules isolées de rein était capable de distinguer les cancérogènes rénaux génotoxiques des cancérogènes rénaux épigénétiques alors que la cytotoxicité ne s'est pas révélée être un facteur interférant : les 5 cancérogènes rénaux agissant selon différents mécanismes génotoxiques ont été clairement détectés directement ou indirectement dans le cas du cis-platine, alors que les cancérogènes rénaux épigénétiques et les produits néphrotoxiques non-cancérogènes se sont révélés clairement négatifs. La réponse positive observée sur l'acide nitrilotriacétique, classé comme cancérogène chez le rongeur agissant par un mécanisme non génotoxique, pourrait être liée à la déprivation en cations divalents (incluant les ions Ca2+) au niveau des cellules du tractus urinaire menant à un effet clastogène local et indirect ce qui suggère l'existence d'une dose seuil au-delà de laquelle des événements générant l'apparition de tumeurs seront observés. En revanche, l'effet néphrocancérogène du tris-chloro-éthyl-phosphate est lié à son activité génotoxique intrinsèque et non à des phénomènes épigénétiques comme cela est décrit dans la littérature. Enfin, la génotoxicité organospécificique du Chloracétal C5, suspecté d'être responsable d'un excès de cancers du rein au sein de la population salariée d'une usine chimique, démontrée in vitro a été confirmée in vivo par le test des comètes. En conclusion, le test des comètes in vivo a montré une grande sensibilité et une bonne spécificité ce qui permet de l'utiliser comme outil pour évaluer le potentiel génotoxique d'un produit lorsque des changements néoplasiques/prénéoplasique ont lieu consécutivement à des traitements subchroniques ou chroniques afin de déterminer le rôle de la génotoxicité dans l'induction de tumeur, et/ou d'en étudier l'organospécificité.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

From Basic Research to New Tools and Challenges for the Genotoxicity Testing of Nanomaterials

Genotoxicity is one of the key endpoints investigated as early as possible before marketing a product [...

Porous Maltodextrin-Based Nanoparticles: A Safe Delivery System for Nasal Vaccines

Vaccination faces limitations, and delivery systems additionally appear to have potential as tools to trigger protective immune responses against diseases. The nanoparticles studied are cationic maltodextrin-based nanoparticles with an anionic phospholipid core (NPL); they are a promising antigen delivery system, and their efficacy as drug vectors against complex diseases such as toxoplasmosis has already been demonstrated. Cationic compounds are generally described as toxic; therefore, it is of interest to evaluate the behavior of these NPL in vitro and in vivo. Here, we studied the in vitro toxicity (cytotoxicity and ROS induction in intestinal and airway epithelial cell lines) and the in vivo tolerability and genotoxicity of these nanoparticles administered by the nasal route to a rodent model. In vitro, these NPL were not cytotoxic and did not induce any ROS production. In vivo, even at very large doses (1000 times the expected human dose), no adverse effect and no genotoxicity were observed in lungs, stomach, colon, or liver. This study shows that these NPL can be safely used

Comparison of In Vitro Endocrine Activity of Phthalates and Alternative Plasticizers

Because of the deleterious effects of phthalates, regulations have been taken to decrease their use, and the needs for alternatives are increasing. Due to the concerns about the endocrine-disrupting properties of phthalates, it was deemed necessary to particularly investigate these effects for potential substitutes. In this study, we compared the in vitro endocrine activity of several already used potential alternative plasticizers (DEHT, DINCH, and TOTM) or new substitutes (POLYSORB® isosorbide and POLYSORB® ID 46) to one of 2 phthalates, DEHP and DINP. Effects of these chemicals on 3 common mechanisms of endocrine disruption, i.e., interaction with estrogen receptors (ER), androgen receptors (AR), or steroidogenesis, were studied using extensively used in vitro methods. In the E-Screen assay, only DEHP moderately induced MCF-7 cell proliferation; none of the other tested substances were estrogenic or antiestrogenic. No androgenic or antiandrogenic activity in MDA-kb2 cells was shown for any of the tested phthalates or alternatives. On the other hand, both DEHP and DINP, as well as DEHT, DINCH, and TOTM, disrupted steroidogenesis in the H295R assay, mainly by inducing an increase in estradiol synthesis; no such effect was observed for POLYSORB® isosorbide and POLYSORB® ID 46