Towards a nanospecific approach for risk assessment.

In the current paper, a new strategy for risk assessment of nanomaterials is described, which builds upon previous project outcomes and is developed within the FP7 NANoREG project. NANoREG has the aim to develop, for the long term, new testing strategies adapted to a high number of nanomaterials where many factors can affect their environmental and health impact. In the proposed risk assessment strategy, approaches for (Quantitative) Structure Activity Relationships ((Q)SARs), grouping and read-across are integrated and expanded to guide the user how to prioritise those nanomaterial applications that may lead to high risks for human health. Furthermore, those aspects of exposure, kinetics and hazard assessment that are most likely to be influenced by the nanospecific properties of the material under assessment are identified. These aspects are summarised in six elements, which play a key role in the strategy: exposure potential, dissolution, nanomaterial transformation, accumulation, genotoxicity and immunotoxicity. With the current approach it is possible to identify those situations where the use of nanospecific grouping, read-across and (Q)SAR tools is likely to become feasible in the future, and to point towards the generation of the type of data that is needed for scientific justification, which may lead to regulatory acceptance of nanospecific applications of these tools.

Human exposure to conventional and nanoparticle--containing sprays-a critical review.

The release of pesticides from conventional spray products has been investigated in depth, and suitable analytical techniques detecting the mass of the released substances are available. In contrast, nanoparticle-containing sprays are less studied, although they are perceived as critical for consumers because inhalation exposure can occur to potentially toxic nanoparticles. A few recent studies presented analytical concepts for exposure experiments and generated data for exposure assessment. This study attempts to review and compare the current approaches to characterize nanosprays and to identify challenges for future research. Furthermore, experimental setups used for exposure assessment from conventional sprays are reviewed and compared to setups used for nanoparticle-containing sprays. National and international norms dealing with nanoparticle characterization, spray characterization and exposure are inspected with regard to their usefulness for standardizing exposure assessment. Different approaches in the field of exposure modeling are reviewed and compared. The conclusion is that due to largely varying experimental setups to date exposure values for nanosprays are difficult to compare. All studies are only conducted with a limited set of sprays, and no systematic evaluation of the study conditions is available. A suitable set of experimental setups as well as minimum reporting requirements should be agreed upon to enable the systematic evaluation of consumer sprays in the future. Indispensable features of such experimental setups are developed in this review.

Assessment of the risk of respiratory sensitization from fragrance allergens released by air fresheners.

Consumers using air fresheners are exposed to the emitted ingredients, including fragrances, via the respiratory tract. Several fragrances are known skin sensitizers, but it is unknown whether inhalation exposure to these chemicals can induce respiratory sensitization. Effects on the immune system were assessed by testing a selection of five fragrance allergens in the respiratory local lymph node assay (LLNA). The probability and extent of exposure were assessed by measuring concentrations of the 24 known fragrance allergens in 109 air fresheners. It was shown that the most frequently used fragrances in air fresheners were D-limonene and linalool. In the respiratory LLNA, these fragrances were negative. Of the other tested chemicals, only isoeugenol induced a statistically significant increase in cell proliferation. Consumer exposure was assessed in more detail for D-limonene, linalool, and isoeugenol by using exposure modeling tools. It was shown that the most frequently used fragrances in air fresheners, D-limonene, and linalool gave rise to a higher consumer exposure compared with isoeugenol. To evaluate whether the consumer exposure to these fragrances is low or high, these levels were compared with measured air concentrations of diisocyanates, known human respiratory sensitizers. This comparison showed that consumer exposure from air fresheners to D-limonene, linalool, and isoeugenol is considerably lower than occupational exposure to diisocyanates. By combing this knowledge on sensitizing potency with the much lower exposure compared to diisocyanates it seems highly unlikely that isoeugenol can induce respiratory sensitization in consumers using air fresheners.

Considerations on the EU definition of a nanomaterial: science to support policy making.

In recent years, an increasing number of applications and products containing or using nanomaterials have become available. This has raised concerns that some of these materials may introduce new risks for humans or the environment. A clear definition to discriminate nanomaterials from other materials is prerequisite to include provisions for nanomaterials in legislation. In October 2011 the European Commission published the 'Recommendation on the definition of a nanomaterial', primarily intended to provide unambiguous criteria to identify materials for which special regulatory provisions might apply, but also to promote consistency on the interpretation of the term 'nanomaterial'. In this paper, the current status of various regulatory frameworks of the European Union with regard to nanomaterials is described, and major issues relevant for regulation of nanomaterials are discussed. This will contribute to better understanding the implications of the choices policy makers have to make in further regulation of nanomaterials. Potential issues that need to be addressed and areas of research in which science can contribute are indicated. These issues include awareness on situations in which nano-related risks may occur for materials that fall outside the definition, guidance and further development of measurement techniques, and dealing with changes during the life cycle.

Review of health safety aspects of nanotechnologies in food production.

Due to new, previously unknown, properties attributed to engineered nanoparticles many new products are introduced in the agro-food area. Nanotechnologies cover many aspects, such as disease treatment, food security, new materials for pathogen detection, packaging materials and delivery systems. As with most new and evolving technologies, potential benefits are emphasized, while little is known on safety of the application of nanotechnologies in the agro-food sector. This review gives an overview of scientific issues that need to be addressed with priority in order to improve the risk assessment for nanoparticles in food. The following research topics are considered to contribute pivotally to risk assessment of nanotechnologies and nanoparticles in food products. Set a definition for NPs to facilitate regulatory discussions, prioritization of research and exchange of study results. Develop analytical tools for the characterization of nanoparticles in complex biological matrices like food. Establish relevant dose metrics for nanoparticles used for both interpretation of scientific studies as well as regulatory frameworks. Search for deviant behavior (kinetics) and novel effects (toxicity) of nanoparticles and assess the validity of currently used test systems following oral exposure. Estimate the consumer exposure to nanoparticles.

Dominant-negative but not gain-of-function effects of a p53.R270H mutation in mouse epithelium tissue after DNA damage.

p53 alterations in human tumors often involve missense mutations that may confer dominant-negative or gain-of-function properties. Dominant-negative effects result in inactivation of wild-type p53 protein in heterozygous mutant cells and as such in a p53 null phenotype. Gain-of-function effects can directly promote tumor development or metastasis through antiapoptotic mechanisms or transcriptional activation of (onco)genes. Here, we show, using conditional mouse technology, that epithelium-specific heterozygous expression of mutant p53 (i.e., the p53.R270H mutation that is equivalent to the human hotspot R273H) results in an increased incidence of spontaneous and UVB-induced skin tumors. Expression of p53.R270H exerted dominant-negative effects on latency, multiplicity, and progression status of UVB-induced but not spontaneous tumors. Surprisingly, gain-of-function properties of p53.R270H were not detected in skin epithelium. Apparently, dominant-negative and gain-of-function effects of mutant p53 are highly tissue specific and become most manifest upon stabilization of p53 after DNA damage.

Tissue specific mutagenic and carcinogenic responses in NER defective mouse models.

Several mouse models with defects in genes encoding components of the nucleotide excision repair (NER) pathway have been developed. In NER two different sub-pathways are known, i.e. transcription-coupled repair (TC-NER) and global-genome repair (GG-NER). A defect in one particular NER protein can lead to a (partial) defect in GG-NER, TC-NER or both. GG-NER defects in mice predispose to cancer, both spontaneous as well as UV-induced. As such these models (Xpa, Xpc and Xpe) recapitulate the human xeroderma pigmentosum (XP) syndrome. Defects in TC-NER in humans are associated with Cockayne syndrome (CS), a disease not linked to tumor development. Mice with TC-NER defects (Csa and Csb) are - except for the skin - not susceptible to develop (carcinogen-induced) tumors. Some NER factors, i.e. XPB, XPD, XPF, XPG and ERCC1 have functions outside NER, like transcription initiation and inter-strand crosslink repair. Deficiencies in these processes in mice lead to very severe phenotypes, like trichothiodystrophy (TTD) or a combination of XP and CS. In most cases these animals have a (very) short life span, display segmental progeria, but do not develop tumors. Here we will overview the available NER-related mouse models and will discuss their phenotypes in terms of (chemical-induced) tissue-specific tumor development, mutagenesis and premature aging features.

Mice expressing a mammary gland-specific R270H mutation in the p53 tumor suppressor gene mimic human breast cancer development.

The tumor suppressor gene p53 has an apparent role in breast tumor development in humans, as approximately 30% of sporadic tumors acquire p53 mutations and Li-Fraumeni syndrome patients carrying germ line p53 mutations frequently develop breast tumors at early age. In the present study, conditional expression of a targeted mutation is used to analyze the role of the human R273H tumor-associated hotspot mutation in p53 in mammary gland tumorigenesis. Heterozygous p53(R270H/+)WAPCre mice (with mammary gland-specific expression of the p53.R270H mutation, equivalent to human R273H, at physiologic levels) develop mammary tumors at high frequency, indicating that the R270H mutation predisposes for mammary gland tumor development and acts in a dominant-negative manner in early stages of tumorigenesis. Spontaneous tumor development in these mice is further accelerated by 7,12-dimethylbenz(a)anthracene (DMBA) treatment at young age. The majority of spontaneous and DMBA-induced carcinomas and sarcomas from p53(R270H/+)WAPCre mice is estrogen receptor alpha positive, and expression profiles of genes also implicated in human breast cancer appear similarly altered. As such, p53(R270H/+)WAPCre mice provide a well-suited model system to study the role of p53 in breast tumorigenesis and the responsiveness of mammary gland tumors to chemotherapeutics.

Accelerated aging pathology in ad libitum fed Xpd(TTD) mice is accompanied by features suggestive of caloric restriction.

Trichothiodystrophy (TTD) patients with a mutation in the XPD gene of nucleotide excision repair (NER) have a short life span and show various features of premature aging, thereby linking DNA damage to the aging process. Xpd(TTD) mutant mice share many features with TTD patients, including a shorter life span, accompanied by a segmental progeroid phenotype. Here we report new pathology features supportive to the premature aging phenotype of Xpd(TTD) mice. Strikingly, accelerated aging pathology is accompanied by signs suggestive of caloric restriction (CR), a condition usually linked to retardation of age-related pathology and life extension. Accelerated aging symptoms in Xpd(TTD) mice are most likely due to accumulation of endogenously generated DNA damage and compromised transcription leading to cell death, whereas CR symptoms may reflect the need of Xpd(TTD) mice to reduce metabolism (ROS production) in an attempt to extend their life span. Our current findings in Xpd(TTD) mice further strengthen the link between DNA damage, repair and aging.

Transgenic and knockout mice for DNA repair functions in carcinogenesis and mutagenesis.

Genetically modified mouse models with defects in DNA repair pathways, especially in nucleotide excision repair (NER) and mismatch repair (MMR), are powerful tools to study processes like carcinogenesis and mutagenesis. The use of mutant mice in these studies has many advantages over using normal wild type mice with respect to costs, number of animals, predictive value towards carcinogenic compounds and the duration of study. Short-term carcinogenicity assays still require considerable number of animals and extensive pathological analyses. Therefore, alternatives demanding less animals and shorter exposure times would be desirable. In this respect, one approach could be the use of transgenic mice harbouring marker genes, that can easily detect mutagenic features of carcinogenic compounds, especially when such models are in a DNA repair deficient background. Here, we review the progress made in the development and use of DNA repair deficient mouse models as replacements for long-term cancer assays and discuss the applicability of enhanced gene mutant frequencies as early indicators of tumourigenesis. Although promising models exist, there is still a need for more universally responding and highly sensitive mouse models, since it is likely that non-genotoxic carcinogens will go undetected in a DNA repair deficient mouse. One attractive candidate mouse model, having a presumptive broad detective range, is the Xpa/p53 mutant mouse model, which will be discussed in more detail.

Analysis of the occupational, consumer and environmental exposure to engineered nanomaterials used in 10 technology sectors.

Humans and the environment can come into contact with nanomaterials through a wide range of applications during all stages of the life cycle of nanoproducts. The aim of this commentary is to present an assessment of the potential for exposure and thus identify possible environmental, health and safety (EHS) issues for nanomaterials used in 10 technology sectors. We analysed all life cycle stages with regard to potential for exposure of workers, consumers/patients, and the environment. A wide variety of nanomaterials are used of which many have negligible potential for exposure, while others have medium or even high potential for exposure. Based on the likelihood of exposure, it appears that in general most attention should be paid to the agrifood, chemistry/materials, textiles and health sectors; and less to the information and communication technology (ICT), security and energy sectors. Toxicity and exposure are both important; however, the EHS impact of nanomaterials is always dependent on their particular use.

Broad segmental progeroid changes in short-lived Ercc1(-/Δ7) mice.

Genome maintenance is considered a prime longevity assurance mechanism as apparent from many progeroid human syndromes that are caused by genome maintenance defects. The ERCC1 protein is involved in three genome maintenance systems: nucleotide excision repair, interstrand cross-link repair, and homologous recombination. Here we describe in-life and post-mortem observations for a hypomorphic Ercc1 variant, Ercc1(-/Δ7), which is hemizygous for a single truncated Ercc1 allele, encoding a protein lacking the last seven amino acids. Ercc1(-/Δ7) mice were much smaller and median life span was markedly reduced compared to wild-type siblings: 20 and 118 weeks, respectively. Multiple signs and symptoms of aging were found to occur at an accelerated rate in the Ercc1(-/Δ7) mice as compared to wild-type controls, including a decline in weight of both whole body and various organs, numerous histopathological lesions, and immune parameters. Together they define a segmental progeroid phenotype of the Ercc1(-/Δ7) mouse model.

Mouse models for xeroderma pigmentosum group A and group C show divergent cancer phenotypes.

The accumulation of DNA damage is a slow but hazardous phenomenon that may lead to cell death, accelerated aging, and cancer. One of the most versatile defense mechanisms against the accumulation of DNA damage is nucleotide excision repair, in which, among others, the Xeroderma pigmentosum group C (XPC) and group A (XPA) proteins are involved. To elucidate differences in the functions of these two proteins, comprehensive survival studies with Xpa(-/-), Xpc(-/-) and wild-type control female mice in a pure C57BL/6J background were done. The median survival of Xpc(-/-) mice showed a significant decrease, whereas the median survival of Xpa(-/-) mice did not. Strikingly, Xpa(-/-) and Xpc(-/-) mice also showed a phenotypical difference in terms of tumor spectrum. Xpc(-/-) mice displayed a significant increase in lung tumors and a trend toward increased liver tumors compared with Xpa-deficient or wild-type mice. Xpa(-/-) mice showed a significant elevation in liver tumors. Additionally, Xpc-deficient mice exhibited a strong increase in mutant frequency in lung compared with Xpa(-/-) mice, whereas in both models mutant frequency is increased in liver. Our in vitro data displayed an elevated sensitivity to oxygen in Xpc(-/-) in mouse embryonic fibroblasts (MEF) when compared with Xpa(-/-) and wild-type fibroblasts. We believe that XPC plays a role in the removal of oxidative DNA damage and that, therefore, Xpc(-/-) mice display a significant increase in lung tumors and a significant elevation in mutant frequency in lung, and Xpc-deficient MEFs show greater sensitivity to oxygen when compared with Xpa(-/-) and wild-type mice.

Chemical carcinogens induce varying patterns of LOH in mouse T-lymphocytes.

We have shown previously that a wide range of mutagenic carcinogens are capable of inducing loss of heterozygosity (LOH) at the endogenous Aprt locus in mouse splenic lymphocytes. To investigate whether LOH might be caused by a single common mechanism, we set out to determine the extent of LOH by microsatellite analysis along (the Aprt gene containing) mouse chromosome 8. Aprt+/- hybrid B6C3F1 mice were treated with mutagens that induce different classes of DNA lesions, i.e. bulky DNA adducts, DNA methylation, DNA inter-strand crosslinks or DNA strand breaks. Aprt mutant frequencies (MF) in this C57Bl/6-C3H hybrid background were significantly reduced for mitomycin C (MMC) and methylmethanesulfonate (MMS) in comparison with MF in C57Bl/6 background, suggesting either enhanced repair or reduced formation of MMC- or MMS-induced mutagenic lesions in a hybrid B6C3F1 background. In contrast, Aprt MF after dimethylbenz[a]anthracene (DMBA), methylnitrosurea (MNU) and etoposide treatment were similar in both genetic backgrounds. Microsatellite analysis of Aprt mutant clones indicated a dominant role for mitotic recombination (MR) in generating spontaneous, DMBA- and etoposide-induced LOH at APRT: However, over 80% of the MMC-induced Aprt LOH mutants had lost heterozygosity for all markers tested, suggesting that either the crossover points were located close to the centromere or that these mutants arose by chromosome loss and duplication of the remaining chromosome 8. A substantial fraction (40%) of MNU-induced Aprt mutants had lost the wild-type Aprt allele, but had retained heterozygosity at all polymorphic markers tested at chromosome 8 indicating an important role for deletions in LOH formation by MNU. Patterns of MR differed quite dramatically for the various chemical mutagens tested, suggesting different mechanisms to be involved in inducing recombination between homologous chromosomes. In addition, non-random adduct formation and repair between chromosomal regions, i.e. heterochromatin versus euchromatin, may contribute to a non-random distribution of recombinational crossover points.