Towards an alternative testing strategy for nanomaterials used in nanomedicine: lessons from NanoTEST.

In spite of recent advances in describing the health outcomes of exposure to nanoparticles (NPs), it still remains unclear how exactly NPs interact with their cellular targets. Size, surface, mass, geometry, and composition may all play a beneficial role as well as causing toxicity. Concerns of scientists, politicians and the public about potential health hazards associated with NPs need to be answered. With the variety of exposure routes available, there is potential for NPs to reach every organ in the body but we know little about the impact this might have. The main objective of the FP7 NanoTEST project ( www.nanotest-fp7.eu ) was a better understanding of mechanisms of interactions of NPs employed in nanomedicine with cells, tissues and organs and to address critical issues relating to toxicity testing especially with respect to alternatives to tests on animals. Here we describe an approach towards alternative testing strategies for hazard and risk assessment of nanomaterials, highlighting the adaptation of standard methods demanded by the special physicochemical features of nanomaterials and bioavailability studies. The work has assessed a broad range of toxicity tests, cell models and NP types and concentrations taking into account the inherent impact of NP properties and the effects of changes in experimental conditions using well-characterized NPs. The results of the studies have been used to generate recommendations for a suitable and robust testing strategy which can be applied to new medical NPs as they are developed

Interobserver Variation in Canine Hip Dysplasia Evaluation

Canine hip dysplasia (CHD) is a common disease representing an important problem for many dog breeds worldwide. The screening for CHD and breeding programs have been ongoing for many decades but the incidence of disease have failed to be reduced to the expected level. The early diagnosis of CHD is paramount in order to facilitate the early management strategies and to prevent the breeding of the affected individuals. Generally in this area, the emphasis is placed on the radiographic evaluation process, however this is partly a subjective process suggested to be influenced by the experience of the observers. This study was designed to evaluate the interobserver agreement in CHD evaluation based on the Federation Cynologique International system (FCI system). Ten original radiographs were sent to five different groups of observers, from students to certified veterinarians. They were asked to evaluate the ventro-dorsal radiographs according to the FCI system which is the most common system used in Europe to give the final grades (A, B, C, D, E). The grades were converted to numbers and the data were analysed using a one-way ANOVA test. The results showed that only in 20% of the cases, the interobserver agreement was statistically higher in the group of the most experienced observers when compared to the less experienced group of observers. This means that the level of experience does not always lead to a higher agreement. This could be a problem of widespread objective evaluations of CHD. In addition, there are several different systems of evaluation used in various countries. It is necessary to understand the intention of dog owners, who when buying a dog may be planning its first breeding. Therefore, the “correct” or “incorrect” assessment of the CHD radiographs may not always result in the elimination of affected individuals. We do not know exactly the situation of the development of the hip in some breeds, because scrutineers are receiving only “negative radiographs” for evaluation. Many owners are very educated about hip and elbow dysplasia. It is a time to tell the scientific truth

Spinal cord haemangiosarcoma in one dog - Case report

A 5-year-old intact female Shih Tzu was presented with acute onset of hind leg paralysis. The neurologic examination revealed severe T3-L3 myelopathy. The differential diagnoses included degenerative, anomalous, traumatic, inflammatory, vascular, metabolic, and neoplastic changes. The results of the paraclinical examinations and diagnostic imaging narrowed the list of differential diagnoses and, along with the patient's deteriorating condition, led to the owner's decision to euthanise the dog. The histologic findings of the spinal cord specimens indicated a tumour of the blood vessels formed by the proliferation of endothelial cells, which may present as either capillary or cavernous structures. In this case, the tumour was a capillary-type haemangiosarcoma. The primary site of proliferation could not be determined in this case because no mass formation was noted while performing the necropsy

DNA repair and cyclin D1 polymorphisms and styrene-induced genotoxicity and immunotoxicity.

1-SO-adenine DNA adducts, DNA single-strand breaks (SBs), chromosomal aberrations (CAs), mutant frequency (MF) at the HPRT gene, and immune parameters (hematological and of humoral immunity) were studied in styrene-exposed human subjects and controls. Results were correlated with genetic polymorphisms in DNA repair genes (XPD, exon 23, XPG, exon 15, XPC, exon 15, XRCC1, exon 10, XRCC3, exon 7) and cell cycle gene cyclin D1. Results for biomarkers of genotoxicity after stratification for the different DNA repair genetic polymorphisms showed that the polymorphism in exon 23 of the XPD gene modulates levels of chromosomal and DNA damage, HPRT MF, and moderately affects DNA adduct levels. The highest levels of biomarkers were associated with the wild-type homozygous AA genotype. The exposed individuals with the wild-type GG genotype for XRCC1 gene exhibited the lowest CA frequencies, compared to those with an A allele (P < 0.05). Cyclin D1 polymorphism seems to modulate the number of leukocytes and lymphocytes in the analyzed subjects. The number of eosinophiles was positively associated with XPD variant C allele and negatively with XRCC1 variant A allele (P < 0.05) and XPC variant C allele (P < 0.05). Immunoglobulin IgA was positively associated with an XRCC3 variant T allele (P < 0.01) and negatively with XPC variant C allele (P < 0.05). Both C3- and C4-complement components were lower in individuals with XRCC3 CT (P < 0.05) and TT genotypes (P < 0.01). Adhesion molecules sL-selectin and sICAM-1 were associated with XPC genotype (P < 0.05). Individual susceptibility may be reflected in genotoxic and immunotoxic responses to environmental and occupational exposures to xenobiotics

Cytogenetic markers, DNA single-strand breaks, urinary metabolites, and DNA repair rates in styrene-exposed lamination workers

The effect of occupational exposure to styrene on frequencies of chromosomal aberrations and binucleated cells with micronuclei and on single-strand break levels in peripheral blood lymphocytes was studied in 86 reinforced plastic workers and 42 control individuals (including 16 maintenance workers with intermittent, low-dose exposure). In these individuals, the irradiationspecific DNA repair rates and the repair rates of 8-oxoguanines were investigated. We assessed the exposure by measuring the concentrations of styrene in air and in blood and of mandelic acid, phenylglyoxylic acid, 4-vinyl phenol conjugates and regioisomeric phenyl hydroxyethyl mercapturic acids in urine. All these parameters correlated with one another. No clear relationship was found between the styrene exposure and the frequencies of chromosomal aberrations. Binucleated cells with micronuclei were moderately related to the parameters of styrene exposure. We found a negative correlation between all exposure parameters and single-strand breaks. The positive correlation between exposure parameters and DNA repair rates suggests that particular DNA repair pathways may be induced by styrene exposure

Coating-dependent induction of cytotoxicity and genotoxicity of iron oxide nanoparticles

Surface coatings of nanoparticles (NPs) are known to influence advantageous features of NPs as well as potential toxicity. Iron oxide (Fe3O4) NPs are applied for both medical diagnostics and targeted drug delivery. We investigated the potential cytotoxicity and genotoxicity of uncoated iron oxide (U-Fe3O4) NPs in comparison with oleate-coated iron oxide (OC-Fe3O4) NPs. Testing was performed in vitro in human lymphoblastoid TK6 cells and in primary human blood cells. For cytotoxicity testing, relative growth activity, trypan blue exclusion, (3)H-thymidine incorporation and cytokinesis-block proliferation index were assessed. Genotoxicity was evaluated by the alkaline comet assay for detection of strand breaks and oxidized purines. Particle characterization was performed in the culture medium. Cellular uptake, morphology and pathology were evaluated by electron microscopy. U-Fe3O4 NPs were found not to be cytotoxic (considering interference of NPs with proliferation test) or genotoxic under our experimental conditions. In contrast, OC-Fe3O4 NPs were cytotoxic in a dose-dependent manner, and also induced DNA damage, indicating genotoxic potential. Intrinsic properties of sodium oleate were excluded as a cause of the toxic effect. Electron microscopy data were consistent with the cytotoxicity results. Coating clearly changed the behaviour and cellular uptake of the NPs, inducing pathological morphological changes in the cells

Ninety-day oral toxicity studies on two genetically modified maize MON810 varieties in Wistar Han RCC rats (EU 7th Framework Programme project GRACE)

The GMO Risk Assessment and Communication of Evidence (GRACE; www.grace-fp7.eu) project is funded by the European Commission within the 7th Framework Programme. A key objective of GRACE is to conduct 90-day animal feeding trials, animal studies with an extended time frame as well as analytical, in vitro and in silico studies on genetically modified (GM) maize in order to comparatively evaluate their use in GM plant risk assessment. In the present study, the results of two 90-day feeding trials with two different GM maize MON810 varieties, their near-isogenic non-GM varieties and four additional conventional maize varieties are presented. The feeding trials were performed by taking into account the guidance for such studies published by the EFSA Scientific Committee in 2011 and the OECD Test Guideline 408. The results obtained show that the MON810 maize at a level of up to 33 % in the diet did not induce adverse effects in male and female Wistar Han RCC rats after subchronic exposure, independently of the two different genetic backgrounds of the event