Propuesta de INCOTERM

From the results of extensive in vivo experimental investigations that have been performed to date involving the respiratory system of humans and animals, there is a general recognition that some of the toxic effects of the nanoparticles observed in vitro were more than duplicated under in vivo conditions.

Moreover, because nanoparticles are highly mobile and can easily penetrate through cellular membranes, they do not only affect the original entry site but, through the complex interrelationships and connections between the different organs and tissues of the body, nanoparticles can and do also reach distant sites and affect their functions. In the following, a brief overview of some of the major current papers on the in vivo physiological effects of bioactive nanoparticles is presented, with a particular emphasis on studies that significantly contributed to our understanding of the possible dangers of this new technology.

In a series of most influential papers published by a group headed by Oberdo¨rster, the fate and progress of inhaled ultrafine particles was not only tracked to airway and lung tissues, but also to the most of the major organs of the body, including the gastrointestinal tract, the central nervous system The Future of Nanotechnology in Food Science and Nutrition 171

and the brain (Oberdo¨rster, 2000; Oberdo¨rster et al, 2002, 2004). Thus, inhaled ultrafine carbon or TiO₂ particles were deposited on tissue surfaces of the airway passages and caused lung inflammation. Moreover, the deposited particles were also translocated to epithelial, endothelial and interstitial sites from where they were quickly distributed to extrapulmonary tissues, such as the liver. The route from interstitial site to the blood circulation was probably via the lymphatic channels or directly via the endothelium. In addition to the clearance pathway from the alveolar region, the ultrafine particles could also be cleared from the tracheobronchial region through the mucociliary escalator into the gastrointestinal tract. The possibility thus exists that the ultrafine particles found in the liver, at least in part, were derived from the gastro-intestinal tract, a finding that may have a major bearing on the subject matter of this chapter. This finding also has some experimental supporting evidence from studies in humans and rats with TiO₂ particles (about 150nm or even larger) that showed that when these particles were ingested with food they translocated to the blood and were then taken up by the liver and spleen and other organs (Jani et al, 1994; Bo¨ckmann et al, 2000), even though with some other metal particles this could not be shown to take place to a measurable extent.

In further studies, one of the most significant findings of the Oberdo¨rster group was that the inhaled ultrafine carbon particles were also transmitted through the olfactory mucosa of the nasal region along the olfactory nerve into the olfactory bulb of the central nervous system (Oberdo¨rster et al, 2004). However, it must also be considered that a fraction of the inhaled nano-particles is known to be rapidly transported into the gastrointestinal tract and from there, after transiting the gut wall, to other organs by the blood circula-tion, where these particles can get coated with lipo/apolipoproteins. It is therefore possible that the already-suggested route across the blood–brain barrier by these coated nanoparticles (Kreuter et al, 2002) could have con-tributed, to some degree, to the total amounts of the carbon nanoparticles found in brain tissues. Similar observations were made in rats inhaling aerosols containing the relatively poorly soluble radioactive manganese phosphate (MnHPO₄). In addition to its deposition in the lungs, the manganese was trans-located to the olfactory bulb by the olfactory nerve. Other tissues, such as the liver, kidneys, pancreas and testes also contained significant amounts of manganese (Dorman et al, 2002). Although no measurements were made on gastrointestinal tissues, it can be assumed that, by a similar mechanism to that observed with carbon nanoparticles, it was through the gastrointestinal tract that the manganese was distributed to these other organs. The results also showed that the more soluble the manganese salt was in the experiments the faster its tissue clearance was.

Even though some of the evidence is contradictory, and dependent on the chemical nature of the nanoparticle (Brown et al, 2002; Nemmar et al, 2002), the consensus is that in most instances after the exposure of the aerial passages the nanoparticles can be relatively quickly distributed throughout the body and even breach the blood–brain barrier. Most of these studies were not primarily

aimed at finding out the mechanisms and physiological and immunological consequences of the nanoparticle uptake of the organs, apart from the general observation that lungs will be inflamed owing to the activation of the immune system. However, there has also been some progress in this respect.

Thus, it was recently shown that polluted air containing ultrafine particles may increase biomarkers of inflammation in the brain of intranasally, oval-bumin-sensitized mice (an asthmatic model), such as the proinflammatory cytokines interleukin 1 alpha and tumour necrosis factor alpha and raise the levels of immune-related transcription factor NF-B (Campbell et al, 2005;

Calderon-Garciduenas, Azzarelli et al, 2002).

As polluted air contains relatively high levels of nanoparticle metals these may directly reach the central nervous system through the olfactory route and there directly induce these inflammatory changes. However, it is also possible these effects are caused by systemic soluble inflammatory mediators indirectly after crossing the blood–brain barrier. Ultrafine carbon black particles have also been shown to enhance respiratory syncytial virus-induced airway reactivity, pulmonary inflammation and chemokine expression (Lambert et al, 2003); and in asthmatic patients the harmful effects of exposure to polluted air is also expected to be increased (Chalupa et al, 2004).

Despite some relatively minor differences in conclusions most studies agree that one of the main causes of the harmful responses in the presence of ultrafine particles in the inhaled air is due to the inflammation induced by oxidative stress (Donaldson et al, 2000). Exposure to insoluble carbon nanoparticles (fullerenes) causes oxidative stress even in the brain of some fish species, such as juvenile largemouth bass (Oberdo¨rster, 2004).

Two studies in which SWCNTs, which are probably the most insoluble and least biodegradable materials, were intratracheally instilled in rats (Warheit et al, 2004) or mice (Lam et al, 2004) revealed that once these fibre-like particles reached interstitial spaces in the lungs they produced multifocal granulomas.

These were non-dose-dependent in rats but were dose-dependent and progressively severe with exposure time in mice. The reason for the difference is probably be due to possible differences in the degree to which the individual nanotubes can and do aggregate into nanofibres and nanorope-like structures that have difficulties in tissue penetration. In the work on rats (see Warheit et al, 2004) it was found that the multifocal granulomas consisted of macrophage-like multinucleated cells that surrounded a bolus of black carbon nanotubes. In the study on mice (see Lam et al, 2004) the nanotubes appeared to be more mobile and from the epithelial tissues macrophages could clear them through the mucociliatory escalator into the oesophagus and then the gastrointestinal tract as was also observed with simple carbon black nano-particles. Thus, these results indicated that even these fine nanotubes have sufficient mobility to be distributed in the body and raise legitimate health concerns. However, once they managed to penetrate subepithelial interstitial spaces they became trapped and were therefore difficult to be removed from the lungs. Hat-stacked carbon nanofibres behaved somewhat similarly in the subcutaneous tissue in rats (Yokoyama et al 2005).

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