Health consequences of exposure to aircraft contaminated air and fume events: a narrative review and medical protocol for the investigation of exposed aircrew and passengers.

Thermally degraded engine oil and hydraulic fluid fumes contaminating aircraft cabin air conditioning systems have been well documented since the 1950s. Whilst organophosphates have been the main subject of interest, oil and hydraulic fumes in the air supply also contain ultrafine particles, numerous volatile organic hydrocarbons and thermally degraded products. We review the literature on the effects of fume events on aircrew health. Inhalation of these potentially toxic fumes is increasingly recognised to cause acute and long-term neurological, respiratory, cardiological and other symptoms. Cumulative exposure to regular small doses of toxic fumes is potentially damaging to health and may be exacerbated by a single higher-level exposure. Assessment is complex because of the limitations of considering the toxicity of individual substances in complex heated mixtures.There is a need for a systematic and consistent approach to diagnosis and treatment of persons who have been exposed to toxic fumes in aircraft cabins. The medical protocol presented in this paper has been written by internationally recognised experts and presents a consensus approach to the recognition, investigation and management of persons suffering from the toxic effects of inhaling thermally degraded engine oil and other fluids contaminating the air conditioning systems in aircraft, and includes actions and investigations for in-flight, immediately post-flight and late subsequent follow up.

From single cells to tissues: interactions between the matrix and human breast cells in real time.

BACKGROUND: Mammary gland morphogenesis involves ductal elongation, branching, and budding. All of these processes are mediated by stroma--epithelium interactions. Biomechanical factors, such as matrix stiffness, have been established as important factors in these interactions. For example, epithelial cells fail to form normal acinar structures in vitro in 3D gels that exceed the stiffness of a normal mammary gland. Additionally, heterogeneity in the spatial distribution of acini and ducts within individual collagen gels suggests that local organization of the matrix may guide morphogenesis. Here, we quantified the effects of both bulk material stiffness and local collagen fiber arrangement on epithelial morphogenesis. RESULTS: The formation of ducts and acini from single cells and the reorganization of the collagen fiber network were quantified using time-lapse confocal microscopy. MCF10A cells organized the surrounding collagen fibers during the first twelve hours after seeding. Collagen fiber density and alignment relative to the epithelial surface significantly increased within the first twelve hours and were a major influence in the shaping of the mammary epithelium. The addition of Matrigel to the collagen fiber network impaired cell-mediated reorganization of the matrix and increased the probability of spheroidal acini rather than branching ducts. The mechanical anisotropy created by regions of highly aligned collagen fibers facilitated elongation and branching, which was significantly correlated with fiber organization. In contrast, changes in bulk stiffness were not a strong predictor of this epithelial morphology. CONCLUSIONS: Localized regions of collagen fiber alignment are required for ductal elongation and branching suggesting the importance of local mechanical anisotropy in mammary epithelial morphogenesis. Similar principles may govern the morphology of branching and budding in other tissues and organs.

Toxicology of nanoparticles.

While nanotechnology and the production of nanoparticles are growing exponentially, research into the toxicological impact and possible hazard of nanoparticles to human health and the environment is still in its infancy. This review aims to give a comprehensive summary of what is known today about nanoparticle toxicology, the mechanisms at the cellular level, entry routes into the body and possible impacts to public health. Proper characterisation of the nanomaterial, as well as understanding processes happening on the nanoparticle surface when in contact with living systems, is crucial to understand possible toxicological effects. Dose as a key parameter is essential in hazard identification and risk assessment of nanotechnologies. Understanding nanoparticle pathways and entry routes into the body requires further research in order to inform policy makers and regulatory bodies about the nanotoxicological potential of certain nanomaterials.

Quantification of nanoparticle uptake by cells using an unbiased sampling method and electron microscopy.

AIMS: By randomly sampling a known fraction of a pellet of cultured cells, we have accurately estimated the mean number of 50 nm gold nanoparticles accumulated within a single cell. Cellular nanoparticle uptake was measured using a combination of stereological sampling techniques and transmission electron microscopy. MATERIALS & METHODS: Nanoparticles were counted individually and their intracellular location was recorded. Quantifying cell and nanoparticle number by analyzing a known fraction of the sample led to precise estimates of intracellular nanoparticle numbers and their spatial locations on an ultrastructural level. We propose a simple and reliable fractionator design and show its applicability and potential using fibroblast cells exposed to 50-nm gold nanoparticles. RESULTS & CONCLUSION: We demonstrate that this approach is suitable for any electron-dense nanomaterial resolvable by electron microscopy and any convex-shaped cells. In addition, the fractionator concept is flexible enough to be used for spatio-temporal or in vivo studies.

Genotoxicity evaluation of amorphous silica nanoparticles of different sizes using the micronucleus and the plasmid lacZ gene mutation assay.

We investigated the potential of four well-characterized amorphous silica nanoparticles to induce chromosomal aberrations and gene mutations using two in vitro genotoxicity assays. Transmission electron microscopy (TEM) was used to verify the manufacturer's nominal size of 10, 30, 80 and 400 nm which showed actual sizes of 11, 34, 34 and 248 nm, respectively. The 80 (34) nm silica nanoparticles induced chromosomal aberrations in the micronucleus assay using 3T3-L1 mouse fibroblasts and the 30 (34) and 80 (34) nm silica nanoparticles induced gene mutations in mouse embryonic fibroblasts carrying the lacZ reporter gene. TEM imaging demonstrated that the majority of nanoparticles were localized in vacuoles and not in the nucleus of 3T3-L1 cells, indicating that the observed DNA damage was most likely a result of indirect mechanisms. Further studies are needed to reveal these mechanisms and to determine the biological relevance of the effects of these particular silica nanoparticles in vivo.

Quantification of nanoparticle uptake by cells using microscopical and analytical techniques.

Quantification of nanoparticles in biological systems (i.e., cells, tissues and organs) is becoming a vital part of nanotoxicological and nanomedical fields. Dose is a key parameter when assessing behavior and any potential risk of nanomaterials. Various techniques for nanoparticle quantification in cells and tissues already exist but will need further development in order to make measurements reliable, reproducible and intercomparable between different techniques. Microscopy allows detection and location of nanoparticles in cells and has been used extensively in recent years to characterize nanoparticles and their pathways in living systems. Besides microscopical techniques (light microscopy and electron microscopy mainly), analytical techniques such as mass spectrometry, an established technique in trace element analysis, have been used in nanoparticle research. Other techniques require 'labeled' particles, fluorescently, radioactively or magnetically. However, these techniques lack spatial resolution and subcellular localization is not possible. To date, only electron microscopy offers the resolving power to determine accumulation of nanoparticles in cells due to its ability to image particles individually. So-called super-resolution light microscopy techniques are emerging to provide sufficient resolution on the light microscopy level to image or 'see' particles as individual particles. Nevertheless, all microscopy techniques require statistically sound sampling strategies in order to provide quantitative results. Stereology is a well-known sampling technique in various areas and, in combination with electron microscopy, proves highly successful with regard to quantification of nanoparticle uptake by cells.

Comet sensitivity in assessing DNA damage and repair in different cell cycle stages.

The comet assay is a sensitive tool for estimation of DNA damage and repair at the cellular level, requiring only a very small number of cells. In comparing the levels of damage or repair in different cell samples, it is possible that small experimental effects could be confounded by different cell cycle states in the samples examined, if sensitivity to DNA damage, and repair capacity, varies with the cell cycle. We assessed this by arresting HeLa cells in various cell cycle stages and then exposing them to ionizing radiation. Unirradiated cells demonstrated significant differences in strand break levels measured by the comet assay (predominantly single-strand breaks) at different cell cycle stages, increasing from G(1) into S and falling again in G(2). Over and above this variation in endogenous strand break levels, a significant difference in susceptibility to breaks induced by 3.5 Gy ionizing radiation was also evident in different cell cycle phases. Levels of induced DNA damage fluctuate throughout the cycle, with cells in G(1) showing slightly lower levels of damage than an asynchronous population. Damage increases as cells progress through S phase before falling again towards the end of S phase and reaching lowest levels in M phase. The results from repair experiments (where cells were allowed to repair for 10 min after exposure to ionizing radiation) also showed differences throughout the cell cycle with G(1)-phase cells apparently being the most efficient at repair and M-phase cells the least efficient. We suggest, therefore, that in experiments where small differences in DNA damage and repair are to be investigated with the comet assay, it may be desirable to arrest cells in a specific stage of the cell cycle or to allow for differential cycle distribution.

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles.

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes. Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering. Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 microg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested. Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.

Fate and effects of CeO2 nanoparticles in aquatic ecotoxicity tests.

Cerium dioxide nanoparticles (CeO2 NPs) are increasingly being used as a catalyst in the automotive industry. Consequently, increasing amounts of CeO2 NPs are expected to enter the environment where their fate in and potential impacts are unknown. In this paper we describe the fate and effects of CeO2 NPs of three different sizes (14, 20, and 29 nm) in aquatic toxicity tests. In each standard test medium (pH 7.4) the CeO2 nanoparticles aggregated (mean aggregate size approximately 400 nm). Four test organisms covering three different trophic levels were investigated, i.e., the unicellular green alga Pseudokirchneriella subcapitata, two crustaceans: Daphnia magna and Thamnocephalus platyurus, and embryos of Danio rerio. No acute toxicity was observed for the two crustaceans and D. rerio embryos, up to test concentrations of 1000, 5000, and 200 mg/L, respectively. In contrast, significant chronic toxicity to P. subcapitata with 10% effect concentrations (EC10s) between 2.6 and 5.4 mg/L was observed. Food shortage resulted in chronic toxicity to D. magna, for wich EC10s of > or = 8.8 and < or = 20.0 mg/L were established. Chronic toxicity was found to increase with decreasing nominal particle diameter and the difference in toxicity could be explained by the difference in surface area. Using the data set, PNEC(aquatic)S > or = 0.052 and < or = 0.108 mg/L were derived. Further experiments were performed to explain the observed toxicity to the most sensitive organism, i.e., P. subcapitata. Toxicity could not be related to a direct effect of dissolved Ce or CeO2 NP uptake or adsorption, nor to an indirect effect of nutrient depletion (by sorption to NPs) or physical light restriction (through shading by the NPs). However, observed clustering of NPs around algal cells may locally cause a direct or indirect effect.

Modelling the comet assay.

The single-cell gel electrophoresis technique or comet assay is widely regarded as a quick and reliable method of analysing DNA damage in individual cells. It has a proven track record from the fields of biomonitoring to nutritional studies. The assay operates by subjecting cells that are fixed in agarose to high salt and detergent lysis, thus removing all the cellular content except the DNA. By relaxing the DNA in an alkaline buffer, strands containing breaks are released from supercoiling. Upon electrophoresis, these strands are pulled out into the agarose, forming a tail which, when stained with a fluorescent dye, can be analysed by fluorescence microscopy. The intensity of this tail reflects the amount of DNA damage sustained. Despite being such an established and widely used assay, there are still many aspects of the comet assay which are not fully understood. The present review looks at how the comet assay is being used, and highlights some of its limitations. The protocol itself varies among laboratories, so results from similar studies may vary. Given such discrepancies, it would be attractive to break the assay into components to generate a mathematical model to investigate specific parameters.

Systematic random sampling of the comet assay.

The comet assay is a technique used to quantify DNA damage and repair at a cellular level. In the assay, cells are embedded in agarose and the cellular content is stripped away leaving only the DNA trapped in an agarose cavity which can then be electrophoresed. The damaged DNA can enter the agarose and migrate while the undamaged DNA cannot and is retained. DNA damage is measured as the proportion of the migratory 'tail' DNA compared to the total DNA in the cell. The fundamental basis of these arbitrary values is obtained in the comet acquisition phase using fluorescence microscopy with a stoichiometric stain in tandem with image analysis software. Current methods deployed in such an acquisition are expected to be both objectively and randomly obtained. In this paper we examine the 'randomness' of the acquisition phase and suggest an alternative method that offers both objective and unbiased comet selection. In order to achieve this, we have adopted a survey sampling approach widely used in stereology, which offers a method of systematic random sampling (SRS). This is desirable as it offers an impartial and reproducible method of comet analysis that can be used both manually or automated. By making use of an unbiased sampling frame and using microscope verniers, we are able to increase the precision of estimates of DNA damage. Results obtained from a multiple-user pooled variation experiment showed that the SRS technique attained a lower variability than that of the traditional approach. The analysis of a single user with repetition experiment showed greater individual variances while not being detrimental to overall averages. This would suggest that the SRS method offers a better reflection of DNA damage for a given slide and also offers better user reproducibility.

Reproducible comet assay of amorphous silica nanoparticles detects no genotoxicity.

Genotoxicity of commercial colloidal and laboratory-synthesized silica nanoparticles was tested using the single cell gel electrophoresis or Comet assay. By using a carefully developed protocol and careful characterization of the nanoparticle dispersions, Comet assays were performed on 3T3-L1 fibroblasts with 3, 6, and 24 h incubations and 4 or 40 microg/ml of silica nanoparticles. No significant genotoxicity was observed for the nanoparticles tested under the conditions described, and results were independently validated in two separate laboratories, showing that in vitro toxicity testing can be quantitatively reproducible.

Kinetics of gold nanoparticles in the human placenta.

We studied the transfer of PEGylated gold nanoparticles through perfused human placenta. In 'once-through' perfusions using 15 and 30nm nanoparticles both maternal and fetal outflows were collected. Recirculating perfusions using 10 or 15nm nanoparticles lasted 6h. The gold concentration in samples was analysed on ICP-MS. The reference compound antipyrine crossed the placenta rapidly, as expected. In open perfusions nanoparticles were detected in maternal but not in fetal outflow, suggesting the lack of placental transfer. During 6h re-circulating perfusions, no particles were detected in fetal circulation. Using transmission electron microscopy (TEM) and silver enhancement, nanoparticles could be visualized in the placental tissue mainly in the trophoblastic cell layer. In in vitro experiments, nanoparticles were taken up by BeWo choriocarcinoma cells and retained inside the cells for an extended period of 48h. In conclusion, PEGylated gold nanoparticles of the size 10-30nm did not cross the perfused human placenta in detectable amounts into the fetal circulation within 6h. Whether PEGylated gold nanoparticles eventually are able to cross placenta and whether nanoparticles affect placental functions needs to be further studied.

Seasonal influences on quantitative changes in sweat-associated anatomy in native and thoroughbred horses.

Stereological techniques were used to assess seasonal influences on morphometric characteristics of hair follicles, sweat and sebaceous glands in abattoir pelts of ponies (PN), thoroughbred (TB) and non-thoroughbred (NTB) horses. Volume density of sweat glands increased significantly from winter (0.061) to summer (0.098) in TB, and showed no change in NTB and a positive tendency in PN. There might be a body surface area : volume effect for sweat gland parameters as PN had smaller values than either TB or NTB, probably attributable to control of heat loss in winter. In summer, the skin remained thick and the volume density of sebaceous glands was increased in NTB, in contrast to TB where both were decreased. It is possible that in summer, sebum has a particular importance in NTB to enhance wicking of sweat through the pelt. TB showed significantly higher volume measurements of sebaceous glands than NTB and PN for winter: sebum has probably a special importance for water-proofing in TB in winter. PN showed no significant seasonal changes in sebaceous glands, but had a thinner summer skin. Winter values for hair follicle volume density between equine groups were similar (TB, NTB 0.066; PN 0.059), as was skin thickness (1.14-1.19 mm). The volume density lowered significantly in summer in TB and NTB. The volume of hair follicles under a unit area of skin surface decreased significantly in TB and nonsignificantly in NTB and PN. The seasonal adaptations of the skin shown here were most pronounced in TB and differed between breeds.

Investigation of the vitamins A and E and beta-carotene content in milk from UK organic and conventional dairy farms.

During a 12-month longitudinal study, bulk-tank milk was collected from organic (n=17) and conventional (n=19) dairy farms in the UK. Milk samples were analysed for vitamin A (retinol), vitamin E (alpha-tocopherol) and beta-carotene content. The farming system type, herd production level and nutritional factors affecting the milk fat vitamin content were investigated by use of mixed model analyses. Conventionally produced milk fat had a higher mean content of vitamin A than organically produced milk fat, although there were no significant differences in the vitamin E or beta-carotene contents between the two types of milk fat. Apart from farming system, other key factors that affected milk fat vitamin content were season, herd yield and concentrate feeding level. Milk vitamin content increased in the summer months and in association with increased concentrate feeding, whilst higher-yielding herds had a lower milk vitamin E and beta-carotene content. Thus, conventional dairy farms in the UK produced milk with a higher vitamin A content, possibly owing to increased vitamin A supplementation in concentrate feeds. However, knowledge of the effects of season, access to fresh grazing or specific silage types and herd production level may also be used by all producers and processors to enhance the vitamin content in milk.

Dairy cow cleanliness and milk quality on organic and conventional farms in the UK.

A subjective cow cleanliness scoring system was validated and used to assess the cleanliness score of dairy cows at different times in the year. A longitudinal study followed a number of farms from summer to winter, and a larger, cross-sectional study assessed a greater number of farms during the housed winter period. The scoring system was demonstrated to be both a repeatable and practical technique to use on-farm and showed that cows become dirtier in the transition from summer grazing to winter housing. Although farming system (organic or conventional) had no effect on cow cleanliness when cows were at grass, when housed in the winter, organic cows were significantly more likely to be cleaner. There was a link between cow cleanliness scores and milk quality, with herds having lower bulk tank somatic cell counts (BTSCC) tending to have a lower (cleaner) median cow cleanliness score; with this relationship strongest for the organic herds. There was no significant link between cleanliness score and Bactoscan (BS) count or clinical mastitis incidence. No major mastitis pathogens were cultured from bulk tank milk samples from the quartile of herds with the cleanest cows in contrast to the quartile of herds with the dirtiest cows, where significant mastitis pathogens were cultured. Based on this study, all farms, especially organic systems, should attempt to keep cows clean as part of subclinical mastitis control.

Synergistic interactions between commonly used food additives in a developmental neurotoxicity test.

Exposure to non-nutritional food additives during the critical development window has been implicated in the induction and severity of behavioral disorders such as attention deficit hyperactivity disorder (ADHD). Although the use of single food additives at their regulated concentrations is believed to be relatively safe in terms of neuronal development, their combined effects remain unclear. We therefore examined the neurotoxic effects of four common food additives in combinations of two (Brilliant Blue and L-glutamic acid, Quinoline Yellow and aspartame) to assess potential interactions. Mouse NB2a neuroblastoma cells were induced to differentiate and grow neurites in the presence of additives. After 24 h, cells were fixed and stained and neurite length measured by light microscopy with computerized image analysis. Neurotoxicity was measured as an inhibition of neurite outgrowth. Two independent models were used to analyze combination effects: effect additivity and dose additivity. Significant synergy was observed between combinations of Brilliant Blue with L-glutamic acid, and Quinoline Yellow with aspartame, in both models. Involvement of N-methyl-D-aspartate (NMDA) receptors in food additive-induced neurite inhibition was assessed with a NMDA antagonist, CNS-1102. L-glutamic acid- and aspartame-induced neurotoxicity was reduced in the presence of CNS-1102; however, the antagonist did not prevent food color-induced neurotoxicity. Theoretical exposure to additives was calculated based on analysis of content in foodstuff, and estimated percentage absorption from the gut. Inhibition of neurite outgrowth was found at concentrations of additives theoretically achievable in plasma by ingestion of a typical snack and drink. In addition, Trypan Blue dye exclusion was used to evaluate the cellular toxicity of food additives on cell viability of NB2a cells; both combinations had a straightforward additive effect on cytotoxicity. These data have implications for the cellular effects of common chemical entities ingested individually and in combination.

Quantitative phenotyping as an efficient means to estimate C-cell number in a knock-in mouse model of MEN2B.

Over the last two decades we have witnessed the generation of hundreds, if not thousands, of lines of genetically altered mice, large numbers of which are being produced in order to model human disease. Given that their creation is still rather technically demanding and labour intensive, the time taken analysing the resultant phenotypes should be such that the maximal amount of information can be gleaned efficiently in an unbiased manner so as to be as close to the 'true' value as possible. In an attempt to characterise a cell-specific phenotype in a genetically defined knock-in mouse model of multiple endocrine neoplasia type 2B (MEN2B) we used a modern, unbiased, stereological approach called the optical fractionator to estimate total cell number in 3-D space. By applying a sampling technique to tissue blocks in a systematic random uniform manner, we demonstrate that the total number of calcitonin-immunoreactive C-cells in the thyroid glands of littermate mice harbouring activating mutations in one or both alleles of ret does not vary significantly (p = 0.46) from an unbiased estimate of 23,000 in wild-type controls; likewise, neither does mean thyroid volume (p = 0.78) when estimated using Cavalieri's principle. We demonstrate that the variation associated with the quantitative phenotyping method is negligible. Using this efficient, unbiased stereological method our results provide new insights into cell number and positioning with consequences for both normal and disease states. In summary, this unbiased stereological technique is conceptually simple, can be applied efficiently, and is pertinent to quantitating a wide variety of cell phenotypes thereby bridging specialisation boundaries. We propose the adoption of this technique to mouse experimental geneticists and recommend its horizontal transmission across all fields within experimental biology.

Modeling the dietary pesticide exposures of young children.

A stepped approach was used to assess the exposures of 1 1/2-4 1/2-year-old children in the United Kingdom to residues of pesticides (dithiocarbamates; phosmet; carbendazim) found in apples and pears. The theoretical possibility that the acute reference dose (ARD) was being exceeded for a particular pesticide/fruit was tested by applying a combination of maximal variability and maximum measured residue relative to an average-body-weight consumer. The actual risk was then quantified by stochastically modeling consumption, from dietary survey data, with individual body weights, against published residue results for 2000-2002 and the variability of residue distribution within batches. The results, expressed as numbers of children per day likely to ingest more than the ARD, were in the range of 10-226.6 children per day, depending upon the pesticide and year of sampling. The implications for regulatory action are discussed.