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.

Mutagenesis by man-made mineral fibres in the lung of rats.

The potential of two asbestos substitute mineral fibres--rock (stone) wool RW1 and glass wool MMVF10--to induce gene mutations, DNA strand breaks, inflammation and oxidative stress has been studied in rats. Male homozygous lamda-lacI transgenic F344 rats were intratracheally instilled with single doses of 1 and 2 mg/animal of fibres or with multiple doses of 2 mg/animal administered weekly on four consecutive weeks (8 mg in total). Exposure to RW1 fibres for 16 weeks significantly increased mutant frequency (MF) in the lung in a dose-dependent manner, while MMVF10 fibres did not exhibit any increase of MF at any dose. RW1 fibres gave a significant increase of MF at a dose of 1 mg. Four weeks after instillation, neither the single nor the multiple doses significantly increased MF for both fibre types. To investigate mechanisms for induction of mutations, other genotoxicity markers and parameters of inflammatory and oxidative damage were determined in relation to MF. A weak correlation of mutagenicity data with other genotoxicity parameters studied was observed. DNA strand breaks as measured by comet assay were increased in alveolar macrophages and lung epithelial cells of RW1 and MMVF10 treated rats. RWl fibres caused more extensive lung inflammation as measured by release of neutrophils into broncho-alveolar lavage fluid than MMVF10 fibres. The effects were observed 16 weeks post-exposure, indicating a persistence of the pathogenic process during the exposure period. Only minor differences in the extent of inflammatory processes were observed between the doses of 2 mg and 4 x 2 mg, suggesting that any threshold for inflammation lies below the dose of 2 mg. With the exception of the highest dose of MMVF10 fibres after 16 weeks of exposure, no significant increase of oxidative damage as measured by levels of malondialdehyde in lung tissue was observed. MMVF10 fibres caused weaker inflammation in the lung of rats and did not exhibit any mutagenic effect. We conclude that a weak but chronic inflammation (more likely than acute inflammation or direct oxidative damage) in the lung tissue of fibre treated rats characterized by moderate influx of inflammatory cells into BAL is probably responsible for the observed mutagenic effect of RW1 fibres.

Mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

In order to get more insight into the mechanism of asbestos-related lung cancer, the mutagenic potential of asbestos was examined in vivo in rat lung. Groups of five transgenic lambda-lacI (Big Blue) rats were intratracheally instilled with single doses of 1 or 2mg, or with four weekly doses of 2mg, per animal of the amosite asbestos. Sixteen weeks after instillation, the mutation frequency was found to be increased in lung DNA by 2-fold at doses of 2 mg (P = 0.035) and of 4 x 2 mg (P = 0.007) amosite. No significant changes were observed after 4 weeks of exposure. In separate experiments, wild-type F344 rats were treated by the same regimen as described above and markers of inflammation, genotoxicity, cell proliferation and lung tissue damage were analysed. Our results indicate a weak but persistent inflammation and cell proliferation which possibly plays a major role in the observed mutagenic effect.

Benzo[a]pyrene-enhanced mutagenesis by asbestos in the lung of lambda-lacI transgenic rats.

To study the suspected mechanism of the interaction between tobacco smoking and asbestos exposure in the modulation of cancer risk, the mutagenic potential of asbestos in combination with the tobacco smoke carcinogen benzo[a]pyrene (B[a]P) was examined in vivo in the rat lung. B[a]P was administered intratracheally in one set of experiments, or by two daily intraperitoneal injections in another set of experiments, to lambdalacI transgenic rats, together with 1, 2 or 4 x 2 mg amosite in one experiment. In the first experiment, the combined action of amosite and B[a]P caused a synergistic (superadditive) increase of mutation frequency in the lung, as compared to groups treated only with asbestos or B[a]P. In the second experiment, i.p. treatment with B[a]P did not significantly alter the mutation frequency induced by amosite, neither after 4 nor after 16 weeks of exposure. The B[a]P-DNA adduct levels were unaffected by amosite co-treatment in both experiments. We assume that the synergistic increase of mutation frequency after intratracheal treatment was due to the mitogenic activities of B[a]P and of amosite. In conclusion, our findings indicate that a weak and delayed mutagenic effect of amosite in rat lung observed in another study was strongly enhanced by the concomitant action of B[a]P. The striking enhancement effect of B[a]P may provide a basis for understanding the suspected synergism of smoking on asbestos carcinogenesis.

Some lung cellular parameters reflecting inflammation after combined inhalation of amosite dust with cigarette smoke by rats.

Cellular changes were followed in lung cell suspensions after 175 day inhalation by rats of concentrations 30 mg/m3 or 60 mg/m3 of amosite asbestos every second day combined with daily exposure to cigarette smoke at 30 mg of total particulate matter (TPM)/m3 air. Concomitantly, lung inflammation was assessed by changes in the bronchoalveolar lavage fluid (BALF). A dose-dependent rise in the BALF inflammatory parameters was found. The rise of the proportion of binucleate (BNC) and multinucleate cells (MNC) in lung cell suspensions was also dose-dependent. It is concluded that, in the experimental assessment of effects of fibrogenic dusts, the number of BNC and of MNC in lung cell suspensions may serve as a useful semiquantitative biomarker of the inflammation.

Evaluation of bronchoalveolar lavage fluid cytotoxic parameters after inhalation exposure to amosite and wollastonite fibrous dusts combined with cigarette smoke.

The aim of this work was to compare the influence of amosite-asbestos and wollastonite fibrous dusts combined with cigarette smoke on chosen cytotoxic parameters of bronchoalveolar lavage fluid (BALF) in rats. Fisher 344 rats inhaled wollastonite or amosite fibrous dusts (60 or 30 mg x m(-3) air) one hour every two days combined with daily breathing of diluted mainstream tobacco smoke (30 mg of TPM x m(-3) air). The experiment lasted 6 months. After sacrifying the animals bronchoalveolar lavage (BAL) was performed and the viability and phagocytic activity of alveolar macrophages (AM), lactate dehydrogenase (LDH) and alkaline phosphatase activity (in the cell-free BALF), acid phosphatase (ACP) and cathepsin D activity (in cell-free BALF and BAL cell suspension) were examined. Exposure to amosite without tobacco smoke significantly decreased the viability of AM and increased the cathepsin D activity in BAL cells. Exposure to wollastonite significantly increased only the cathepsin D activity in BAL cells. Smoking significantly depressed the phagocytic activity of AM and amplified the amosite-induced increase of lysosomal enzyme activities--especially the activity of cathepsin D in BAL cells.

Impact of acute and subchronic asbestos exposure on some parameters of antioxidant defense system and lung tissue injury.

Asbestos fibers have been used in industry for decades. Deleterious effect of asbestos on the lungs has been documented. However, the mechanism of asbestos related diseases has not been fully explained yet. Numerous papers suggest there is a role of reactive oxygen intermediates (ROI) in asbestos-induced lung disease development. The excess ROI produced can be removed from the lungs by enzymatic and nonenzymatic antioxidants. The aim of our study was to compare the levels of antioxidants (ascorbic acid, retinol, alpha-tocopherol, glutathionperoxidase) as well as some markers of lung injury (lipid peroxides, total amount of protein, alkaline phosphatase) in asbestos treated Wistar-rats both 24 hr and 3 months after exposure to those in the controls, and to find out if the changes in antioxidant levels could affect impairment of the lungs. Decreased levels of antioxidants and increased values of lung tissue injury parameters in exposed groups suggest involvement of ROI in the mechanism of asbestos lung disease development, resulting in lung tissue injury, both 24 hr and 3 months after exposure.

Some bronchoalveolar lavage parameters and leukocyte cytokine release in response to intratracheal instillation of short and long asbestos and wollastonite fibres in rats.

We investigated the differences between the lavage parameters -- including tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) release by lavage leukocytes -- in control rats and in animals intratracheally instilled with short and long amosite and wollastonite fibres. These cytokines can play an important role in lung disease development after long-term exposure to some fibrous dusts. Short and long amosite and wollastonite fibres were intratracheally instilled in rats (1 mg/week) for ten weeks while saline was given to controls. To compare the harmful effects of these fibres, the number of leukocytes/ml of bronchoalveolar lavage (BAL), the number of alveolar macrophages (AM) per ml of BAL, AM:granulocyte (GR) ratios in lavage fluid, phagocytic activity and viability of AM, lactate dehydrogenase (LDH), acid phosphatase (AcP), and TNF-alpha and IFN-gamma release by lavage leukocytes were investigated 3 months after the first intratracheal instillation. Compared with the controls, amosite short fibres significantly decreased the numbers of AM/ml BAL, and increased their phagocytic activity and AcP release. Long amosite fibres significantly decreased the numbers of AM/ml BAL, increased the number of granulocytes depressed the phagocytic activity and viability of AM, and significantly decreased the levels of TNF-alpha and IFN-gamma in supernatants of cultured leukocytes. While wollastonite short and long fibre instillation did not significantly influence the parameters studied (except for a significantly increased number of leukocytes/ml BAL in wollastonite long fibres), amosite short and long fibres caused marked differences in these parameters, the long fibres being more effective.

One-year follow-up of the phagocytic activity of leukocytes after exposure of rats to asbestos and basalt fibers.

The phagocytic activity of leukocytes in peripheral blood was investigated after 2, 24, and 48 hr; 1, 2, 4, and 8 weeks; and 6 and 12 months following intraperitoneal administration of asbestos and basalt fibers to Wistar rats. Asbestos and basalt fibers differed in their effects on the parameters studied. Both granulocyte count and phagocytic activity of leukocytes during the 1-year dynamic follow-up in both dust-exposed groups of animals changed in two phases, characterized by the initial stimulation of the acute phase I, followed by the suppression of the parameters in the chronic phase II. Exposure to asbestos and basalt fibers led, in phase II, to impairment of the phagocytic activity of granulocytes. Asbestos fibers also significantly decreased phagocytic activity of monocytes. Exposure to basalt fibers did not affect the phagocytic activity of monocytes in phase II. Results suggest that the monocytic component of leukocytes plays an important role in the development of diseases caused by exposure to fibrous dusts, but basalt fibers have lesser biological effects than asbestos fibers.

The changes of some immunological parameters in subjects exposed to asbestos in dependence on age, duration of exposure, radiological findings and smoking habits.

We followed the changes in some parameters in men exposed to asbestos (to chrysotile and in part to crocidolite). The studied group consisted of exposed workers and retired workers who had been exposed to asbestos in the past. The results were compared with those in the control group of unexposed adults and analysed according to age, duration of exposure, smoking habits and radiological findings. Investigated were: the levels of immunoglobulins IgA, IgG, IgM, complement components C3, C4, alpha-1-antitrypsin and transferrin (humoral immunity) as well as the phagocytic activity of the leukocytes in peripheral blood (natural immunity). The comparison of the results with those in the control group showed that asbestos exposure stimulated markedly IgG and C4 production and to a lesser extent also IgA, IgM, C3 and transferrin production and it suppressed natural immunity. Comparison of the results of the same age categories--control (40.8), with the subgroup of active workers (44.4), showed significantly increased levels of IgA, IgG, IgM, C4 and decreased values of natural immunity in asbestos exposed group. Comparison of different age categories (pensioners--63.2: workers--44.4) showed, that from five parameters in which statistically significant differences were found, four had decreased values in pensioners. Radiological findings suggested, that changes in immunological parameters probably preceded pathologic processes. There were statistically significant differences in the levels of IgM in smokers compared with nonsmokers in the subgroup of workers exposed to asbestos and in the levels of IgG and C4 in the group of workers + pensioners together. It follows from the present study that asbestos exposure modulates the immune response and leads to significant changes of humoral and natural immunity.

[Dynamic studies of the leukocyte phagocytic activity after exposure of rats to asbestos and basalt fibers]. / Dynamische Studien der phagozytären Leukozytenaktivität nach der Exposition von Asbest- und Basaltfasern bei Ratten.

The paper presents the results of the dynamic one-year follow-up of the phagocytic activity of Wistar-rats peripheral blood leukocytes following intraperitoneal administration of asbestos and basalt fibres (Man-Made Mineral Fibres--MMMF). We investigated the phagocytic activity of leukocytes in peripheral blood following intraperitoneal administration of asbestos and basalt fibres to rats 2, 24, 48 h as well as 1, 2, 4, 8 weeks and 6 and 12 months after dosing. We investigated the time dependent of the changes of relative granulocytes count, percentage of phagocytizing cells from leukocytes, percentage of phagocytizing granulocytes and percentage of phagocytizing monocytes. The results of our experiment showed that asbestos and basalt fibres differed in their effects on the parameters studied. Granulocyte count as well as the phagocytic activity of leukocytes during the one-year dynamic follow-up in both dust--exposed groups of animals were found to change in two phases, characterised by the initial stimulation of the acute phase (I), followed by the suppression of the parameters in the chronic phase (II). Exposure to asbestos and basalt fibres led, in phase II, to impairment of the phagocytic activity of granulocytes. Asbestos fibres at the same time significantly decreased also the phagocytic activity of monocytes. Exposure to basalt fibres did not affect the phagocytic activity of monocytes in phase II. It follows from the results of the experiment, that the monocytic component of leukocytes probably plays an important role in the development of diseases caused by exposure to fibrous dusts and basalt fibres have smaller biological effects compared with asbestos fibres.

[Several immunological characteristics of occupational exposure to asbestos and other mineral fibers]. / Untersuchung einiger immunologischer Merkmale bei professioneller Asbestexposition und bei anderen Mineralfasern.

The authors evaluated some immunologic factors in workers exposed to asbestos, basalt and glass fibres and compared the results with a control reference group. The findings indicate that there is a considerable difference between the immunologic reaction of the organism, mainly when the humoral component of the answer is taken into account, in the exposed and in the control group. The same observation was valid, when the asbestos exposed group was compared with the groups exposed to asbestos substitutes (basalt and glass fibres). The immunoglobulins of the classes A, G, M, complement components C3 and C4 were significantly increased, alpha-1-antitrypsin and phagocytic activity of leukocytes in peripheral blood was decreased. Most of the examined indicators in the basalt- and glass fibres exposed groups differed insignificantly from the control group. Transferrin level was not significantly different in any of the examined groups. The results indicate that there is higher tolerability of basalt and glass fibres in the organism compared with asbestos fibres.

The effect of low-level ozone concentrations on the serum levels of immunoglobulins, alpha-1-antitrypsin and transferrin and on the activation of peripheral lymphocytes.

Long-term exposure (2--9 years) to ozone levels exceeding the Czechoslovak value of mean maximum allowable concentration (0.1 mg . m-3) turned out to induce changes in the exposed persons which can be characterized as initial immune disorders. Higher levels of IgG, IgA, IgM, alpha-1-antitrypsin and transferrin detected in comparison to the reference value in the group of ozone-exposed persons and significantly increased alpha-1-antitrypsin and transferrin levels in comparison to the control group of ore miners can be interpreted as a result of changed adaptation mechanisms in the organism. Analysis of the distribution pattern of the frequency of IgA and IgG changes exceeding the standard deviation shows that the group of ozone-exposed subjects exhibits a higher frequency of these changes than the control group of ore miners. However, the concurrently compared relative numbers of increases and decreases in both groups seem to indicate a considerable interindividual variability in the indicators studied.