François Huaux

Absence of Carcinogenic Response to Multiwall Carbon Nanotubes in a 2-Year Bioassay in the Peritoneal Cavity of the Rat

Toxicological investigations of carbon nanotubes have shown that they can induce pulmonary toxicity, and similarities with asbestos fibers have been suggested. We previously reported that multiwall carbon nanotubes (MWCNT) induced lung inflammation, granulomas and fibrotic reactions. The same MWCNT also caused mutations in epithelial cells in vitro and in vivo. These inflammatory and genotoxic activities were related to the presence of defects in the structure of the nanotubes. In view of the strong links between inflammation, mutations and cancer, these observations prompted us to explore the carcinogenic potential of these MWCNT in the peritoneal cavity of rats. The incidence of mesothelioma and other tumors was recorded in three groups of 50 male Wistar rats injected intraperitoneally with a single dose of MWCNT with defects (2 or 20 mg/animal) and MWCNT without defects (20 mg/animal). Two additional groups of 26 rats were used as positive (2 mg UICC crocidolite/animal) and vehicle controls. After 24 months, although crocidolite induced a clear carcinogenic response (34.6% animals with mesothelioma vs. 3.8% in vehicle controls), MWCNT with or without structural defects did not induce mesothelioma in this bioassay (4, 0, or 6%, respectively). The incidence of tumors other than mesothelioma was not significantly increased across the groups. The initial hypothesis of a contrasting carcinogenic activity between MWCNT with and without defects could not be verified in this bioassay. We discuss the possible reasons for this absence of carcinogenic response, including the length of the MWCNT tested (< 1 mum on average), the absence of a sustained inflammatory reaction to MWCNT, and the capacity of these MWCNT to quench free radicals.

Influence of particle surface area on the toxicity of insoluble manganese dioxide dusts

Abstract The objective of this study was to examine the influence of specific surface area on the biological activity of insoluble manganese dioxide (MnO2) particles. The biological responses to various MnO2 dusts with different specific surface area (0.16, 0.5, 17 and 62u2009m2/g) were compared in vitro and in vivo. A mouse peritoneal macrophage model was used to evaluate the in vitro cytotoxic potential of the particles via lactate dehydrogenase (LDH) release. In vivo, the lung inflammatory response was assessed by analysis of bronchoalveolar lavage after intratracheal instillation in mice (LDH activity, protein concentration and cellular recruitment). In both systems, the results show that the amplitude of the response is dependent on the total surface area which is in contact with the biological system, indicating that surface chemistry phenomena are involved in the biological reactivity. Freshly ground particles with a specific surface area of 5u2009m2/g were also examined in vitro. These particles exhibited an enhanced cytotoxic activity, which was almost equivalent to that of 62u2009m2/g particles, indicating that undefined reactive sites produced at the particle surface by mechanical cleavage may also contribute to the toxicity of insoluble particles. We conclude that, when conducting studies to elucidate the effect of particles on the lung, it is important for insoluble particles such as manganese dioxide to consider the administered dose in terms of surface area (e.g.u2009m2/kg) rather than in gravimetric terms (e.g.u2009mg/kg).

SINTERED INDIUM-TIN-OXIDE (ITO) PARTICLES : A NEW PNEUMOTOXIC ENTITY

Indium-Tin-Oxide (ITO) is a sintered mixture of indium- (In(2)O(3)) and tin-oxide (SnO(2)) in a ratio of 90:10 (wt:wt) that is used for the manufacture of LCD screens and related high technology applications. Interstitial pulmonary diseases have recently been reported in workers from ITO producing plants. The present study was conducted to identify experimentally the exact chemical component responsible for this toxicity and to address possible mechanisms of action. The reactivity of respirable ITO particles was compared with that of its single components alone or their unsintered 90:10 mixture (MIX) both in vivo and in vitro. For all endpoints considered, ITO particles behaved as a specific toxic entity. In vivo, after a single pharyngeal administration (2-20 mg per rat), ITO particles induced a strong inflammatory reaction. At day 3, the inflammatory reaction (cell accumulation, LDH and protein in bronchoalveolar lavage fluid) appeared more marked with ITO particles than with each oxide separately or the MIX. This inflammatory reaction persisted and even worsened after 15 days. After 60 days, this inflammation was still present but no significant fibrotic response was observed. The cytotoxicity of ITO was assessed in vitro in lung epithelial cells (RLE) and macrophages (NR8383 cell line). While ITO particles (up to 200 microg/ml) did not affect epithelial cell integrity (LDH release), a strong cytotoxic response was found in macrophages exposed to ITO, but not to its components alone or mixed. ITO particles also induced an increased frequency of micronuclei in type II pneumocytes in vivo but not in RLE in vitro, suggesting the preponderance of a secondary genotoxic mechanism. To address the possible mechanism of ITO toxicity, reactive oxygen species production was assessed by electron paramagnetic resonance spectrometry in an acellular system. Carbon centered radicals (COO-.) and Fenton-like activity were detected in the presence of ITO particles, not with In(2)O(3), SnO(2) alone, or the MIX. Because the unsintered mixture of SnO(2) and In(2)O(3) particles was unable to reproduce the reactivity/toxicity of ITO particles, the sintering process through which SnO(2) molecules are introduced within the crystal structure of In(2)O(3) appears critical to explain the unique toxicological properties of ITO. The inflammatory and genotoxic activities of ITO dust indicate that a strict control of exposure is needed in industrial settings.

Reduction of the ex vivo production of tumor necrosis factor alpha by alveolar phagocytes after administration of coal fly ash and copper smelter dust.

We investigated the effect of intratracheally instilled coal fly ash (FA) and copper smelter dust (CU) on the lung integrity and on the ex vivo release of tumor necrosis factor alpha (TNF-alpha) by alveolar phagocytes. Groups of female NMRI mice received a single intratracheal administration of different particles normalized for the arsenic content (20 micrograms/kg body weight, i.e., 600 ng arsenic/mouse) and the particle load (100 mg/kg body weight, i.e., 3 mg/mouse). Mice received tungsten carbide (WC) alone (100 mg/kg), FA alone (100 mg/kg, i.e., 20 micrograms arsenic/kg), CU mixed with WC (CU, 13.6 mg/kg, i.e., 20 micrograms arsenic/kg; WC, 86.4 mg/kg) and Ca3(AsO4)2 mixed with WC (20 micrograms arsenic/kg; WC, 100 mg/kg). Animals were sacrificed at 1, 6, or 30 d posttreatment and analyzed by bronchoalveolar lavage for total protein (TP) content, inflammatory cell number and type, and TNF-alpha production. Additional mice were studied to evaluate particle retention by measuring total arsenic retention in the lung at appropriate times. Instillation of WC induced a mild and transient (d 1) inflammatory reaction characterized by an increase of TP and an influx of polymorphonuclear leukocytes in the alveolar compartment. Compared to WC, Ca3(AsO4)2 produced a significant increase of TP content in BALF. CU particles caused a severe but transient inflammatory reaction, while a persisting alveolitis (30 d) was observed after treatment with FA. Compared to control saline, a marked inhibition of TNF-alpha release was observed in response to LPS in all groups at d 1. Cytokine production was upregulated in WC- and Ca3(AsO4)1-treated animals after 6 and 30 d, respectively. However, a 90% inhibition of TNF-alpha production was still observed at d 30 after administration of CU and FA. Although arsenic was cleared from the lung tissue 6 d after Ca3(AsO4)2 administration, a significant fraction persisted (10-15% of the arsenic administered) in the lung of CU- and FA-treated mice at d 30. We hypothetize that suppression of TNF-alpha production is dependent upon the slow elimination of the particles and their metal content from the lung.