INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Keywords: air pollution, toxic inhalation

Several epidemiological studies have shown that peaks of urban air pollution are associated with an increased morbidity and mortality. Gaseous pollutants, such as sulfur dioxide, nitrogen oxides, or ozone, probably play some role, but the most consistently observed conclusion of these epidemiological studies is that the excess mortality from air pollution is mainly associated with particulate matter with a diameter below 10 µm (PM₁₀) (1). A striking feature of these observations is that peaks of air pollution not only have respiratory effects, but they also increase cardiovascular morbidity and mortality (4). In fact, more people seem to die from cardiovascular than from pulmonary diseases during episodes of urban air pollution (7). Moreover, a number of recent epidemiological studies lend support to the concept that parameters of cardiovascular function are affected by particulate air pollution (8, 9).

The mechanisms underlying the respiratory and particularly the cardiovascular effects remain unclear (10). Ultrafine particles (UFPs) with diameter  0.1 µm represent a substantial component, in term of particle numbers, in PM₁₀, although they represent a relatively small fraction of the total mass (11). Moreover, several experimental studies have shown that UFPs have marked toxicity compared with larger particles (12, 13). Indeed, UFPs are able to penetrate deeply into the respiratory tract, and they have a larger surface area than the particles of larger size, thus causing a greater inflammatory response (14). The mechanisms responsible for the cardiovascular effects are not understood, and this has even led to some doubt about the causal association between pollution and mortality. One of the hypotheses to explain the observed effects is that the particles cause inflammatory reactions in the lungs, and this leads to the release of mediators, which may influence the heart, coagulation, or other cardiovascular endpoints (15).

An alternative but not necessarily contradictory hypothesis is that the particles translocate from the lungs into the systemic circulation and thus, directly or indirectly, influence hemostasis or cardiovascular integrity. This possibility, which has received scant attention until now, could be particularly applicable to ultrafine particles, which presumably can enter the circulation.

Therefore, the purpose of our study was to evaluate to what extent and how rapidly technetium-99m-labeled ultrafine particles of denatured albumin (^99mTc-albumin) pass into the systemic circulation after intratracheal administration.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Ultrafine Particles

We used colloidal particles of denaturated serum albumin, commercially available in labeling kits for the preparation of "^99mTc-nanocolloid" (Nanocoll, Nycomed Amersham Sorin, Milan, Italy), which is commonly used for diagnostic purposes in nuclear medicine (16, 17). The kit contains 0.5 mg denatured human serum albumin in lyophilized form. After reconstitution with ^99mTc-pertechnetate, more than 95% of the particles have a diameter below 80 nm (Nanocoll, Nycomed Amersham Sorin, last revision 1999). A mass of 0.5 mg denaturated albumin corresponds to 3.5 × 10¹³ particles.

To a Nanocoll labeling vial were successively added 0.5 ml of 0.5 M phosphate buffer (pH 7.4) and 0.5 ml of generator eluate (Ultratechnekow generator, Mallinckrodt Medical, Petten, Holland) containing 18.5 MBq of technetium-99m as sodium pertechnetate. The vial was shaken for a few seconds and maintained at room temperature for 5-10 min.

The proportion of radiolabeled nanocolloid albumin particles and pertechnetate after labeling was determined by thin-layer chromatography (TLC) using silica impregnated glass fiber 13 cm × 1 cm ITLC-SG strips (Gelman Sciences, Ann Arbor, MI) with NaCl 0.9% as the mobile phase. The chromatograms were monitored for radioactivity with a thin-layer radiochromatography scanner equipped with a 2-in. NaI (Tl) scintillation detector coupled to a single-channel analyzer and a Rachel analysis program (version 1.40, Lablogic, Sheffield, UK).

To verify the size of the particles, we have filtered suspension through filters with pore diameter of 100 nm and 200 nm and then performed TLCs on the filtrates.

Intratracheal Instillation of Nanocolloid Albumin Particles

This project has been approved by our institution's ethical committee for animal experimentation. To avoid particle aggregations, the preparations were always sonicated for 15 min and vortexed immediately (< 1 min) before instillation. Male and female hamsters (Pfd Gold, K.U.Leuven, Leuven, Belgium) weighing 120-130 g were anesthetized by an intraperitoneal injection of sodium pentobarbital (60 mg/ kg). The pretracheal zone was shaved and disinfected with ethanol (70%), and the trachea was exposed. Then 200 µl of sterile NaCl 0.9% containing 100 µg of albumin nanocolloid particles, labeled with 3.7 MBq ^99mTc, was instilled in the trachea by means of a 27-gauge needle. At 5, 15, 30, or 60 min following instillation, the animals were killed by decapitation and dissected (n = 3 at each time point). The order of sampling was as follows to avoid contamination: blood flowing from the cervical blood vessels, abdominal organs, brain, lungs (with airways), and heart (after flushing of its blood). The radioactivity in these samples, as well as in the rest of the body, was measured using a gamma-counter (1480W12ARD, Wallac, Turku, Finland), with a correction being made for background radiation; all counts recorded were summed and found to be equal to the dose administered. The nature of the radioactivity in blood after instillation of ^99mTc-albumin nanocolloid particles was checked at each time point (5, 15, 30, and 60 min) by TLC, as described above. This was also done after instillation of 200 µl of free ^99mTc-pertechnetate (3.7 MBq).

To study dose dependence, similar measurements were made 30 min after the intratracheal instillation of 10 µg (n = 3) or 1 µg (n = 3) of nanocolloid albumin particles, radiolabeled in such way that the same amount of radioactivity (3.7 MBq) was administered per animal.

Results are expressed as means with standard deviations. Comparisons between groups were performed by one-way ANOVA.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Labeling Experiments

TLC of the reaction mixture after labeling nanocolloid albumin with ^99mTc showed that the radiolabel stayed at the application point, whereas ^99mTc in the form of pertechnetate migrated with the solvent front. In all ^99mTc-albumin preparations, the amount of free pertechnetate was negligible, indicating a > 99% efficiency of the labeling onto the albumin particles.

Filtration of the preparation showed that 63% of the dose passed through the filter of 100 nm, and more than 95% passed through a filter of 200 nm. The TLC showed only one peak that remained at the application point, thus corresponding to particle-bound radioactivity.

Intratracheal Instillation of Nanocolloid Albumin Particles

Figure 1 shows that 5, 15, 30, and 60 min after intratracheal instillation of 3.7 MBq ^99mTc-albumin there was a single peak of radioactivity, which remained at the application point of the TLC, in blood. In contrast, after the administration of 3.7 MBq free ^99mTc-pertechnetate, there was also a single peak of radioactivity, but it moved with the solvent front (Figure 2).

View larger version (15K): [in this window] [in a new window]   Figure 1.   Radioactivity in blood after intratracheal administration of 100 µg of 99mTc-albumin nanocolloid particles (3.7 MBq) in hamsters. The values are means ± SD (n = 3). The four inserts show the profiles of thin-layer chromatography of blood in one representative animal at each time point. The y-axis represents CPM (counts per minute) × 1000; the x-axis represents distance (in cm) on the chromatogram. Only one peak, corresponding to albumin-bound 99mTc, is observed.

View larger version (12K): [in this window] [in a new window]   Figure 2.   Thin-layer chromatography of blood after intratracheal instillation of free 99mTc-pertechnetate (3.7 MBq) in one representative animal at each time point. The y-axis represents CPM (counts per minute) × 1000; the x-axis represents distance (in cm) on the chromatogram. Only one peak, corresponding to free 99mTc-pertechnetate, is observed.

Figure 1 illustrates the time course of the radioactivity in blood expressed as a percentage of the intratracheally administered activity per gram of blood. The radioactivity decreased from 2.88 ± 0.80% at 5 min to 1.30 ± 0.17% at 15 min, 1.52 ± 0.46% at 30 min, and 0.21 ± 0.06% at 60 min. An additional experiment was carried out in two hamsters killed 30 min after instillation, in which blood was sampled from the abdominal vena cava rather than from the cervical vessels after decapitation. We recorded similar levels of radioactivity (0.97% and 1.18%) as those found at the same time (30 min) in the other experiments, indicating that the levels found were not due to contamination by tracheal contents.

In the lungs, the radioactivity remained stable at 62 ± 3%, 72 ± 5%, 60 ± 4%, and 73 ± 13% of total radioactivity at 5, 15, 30, and 60 min, respectively. The results of liver, heart, spleen, kidneys, and brain are illustrated in Figure 3. The highest levels of radioactivity were found in the liver, with 0.10 ± 0.07% of total radioactivity at 5 min, increasing to 0.23 ± 0.06% at 15 min and an apparent peak of 1.24 ± 0.27% at 30 min, and then decreasing to 0.06 ± 0.06% at 60 min. The radioactivity values recorded in heart were 0.03 ± 0.01%, 0.07 ± 0.04%, 0.22 ± 0.11%, and 0.1 ± 0.03% at 5, 15, 30, and 60 min, respectively. Detectable values were recorded in kidneys, spleen, and brain.

View larger version (11K): [in this window] [in a new window]   Figure 3.   Radioactivity of various organs after intratracheal administration of 100 µg of 99mTc-albumin nanocolloid particles (3.7 MBq) in hamsters. Values are in percentages of the dose per whole organ. The values are means ± SD (n = 3).

Table 1 compares the findings obtained 30 min after intratracheal administration of 100, 10, and 1 µg ^99mTc albumin nanocolloid particles. With all three doses, values of the same relative magnitudes were obtained in blood as well as in organs.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study was initiated as part of an effort to find biological plausibility for the intriguing, but consistent epidemiological association between peaks of particulate air pollution and cardiovascular mortality and morbidity (18). To date, the preferred line of investigation to explain this association has consisted of exploring whether particle-induced pulmonary inflammation leads to systemic effects, for example, through the release of bioactive cytokines in the circulation (15). This line of investigation is not without merit, but it has not been met with much success (19). Here, we have investigated an alternative hypothesis, namely that ultrafine particles pass directly from the lungs into the blood circulation. This possibility has sometimes been evoked by researchers in the field (20, 21) but, to our knowledge, hard evidence for such translocation has not been provided, and data about the extent and kinetics of the passage of inhaled particles into the circulation are even less available. The novel information from our work is that we have established that a substantial proportion of intratracheally instilled particles of less than 80 nm diameter pass rapidly, that is, within 5 min, into the circulation of hamsters.

One of the initial problems of our experiments has been to find ultrafine particles that could be suitably labeled and easily detected in blood. The best option in terms of sensitivity was to use radioactive labeling. Ideally, we would have liked to label relevant pollutant particles such as residual oil fly ash or diesel exhaust, but this was not within our reach. It was also not possible to label ultrafine particles of carbon black, titanium dioxide, or polystyrene, which have been utilized in this context (22). Therefore, we chose to use commercially available nanocolloid albumin particles, which can be easily labeled with ^99mTc for use in diagnostic nuclear medicine. These particles are of the right size (80 nm) to be considered as ultrafine (< 100 nm) and, as they consist of denatured albumin, they offer the advantage of probably causing no injury to the airway epithelium, unlike some of the pollutant particles mentioned above. The intratracheal instillation of particles could also be criticized as being nonphysiological, but this method of delivery has been shown to be a convenient and valid, though admittedly not perfect, mode of administration of foreign compounds to the airways (25). So, although we recognize that the particles chosen for our study and their mode of administration were not entirely satisfactory to investigate the fate of inhaled pollutant particles, we believe that our main findings are not invalidated by these shortcomings. Further work with inhaled pollutant particles will validate our findings.

An important issue in our experiments was to be satisfied that the radioactivity that was detected in blood consisted of particle-bound radioactivity, that is, radioactivity associated with albumin that had crossed the airway-capillary barrier, rather than free radioactivity that had passed as pertechnetate. Various lines of evidence make the latter possibility unlikely. First, thin-layer chromatography of all blood samples showed only one peak of radioactivity at the application point, corresponding to the situation with the freshly prepared ^99mTc-albumin nanocolloid suspension. Conversely, after the intratracheal administration of pure ^99mTc-pertechnetate, no such peak was apparent, thus excluding the possibility that free technetium became bound onto plasma proteins or cellular components. Finally, the apparent progressive concentration of radioactivity in the liver is compatible with an accumulation of particulates by Kupffer cells, as is known to occur with ultrafine gold colloid or polystyrene particles (26). We are, therefore, confident that we have demonstrated the existence of a passage of particle-bound radioactivity from the lungs into the blood.

This passage proved to be rapid and substantial. Radioactivity was detected as soon as 5 min after intratracheal instillation and the proportion of particles that left the lungs amounted to 2.88% of the administered dose per gram of blood. Assuming a total blood volume of 7 ml per 100 g body mass and a specific density of 1.060 for blood, up to 25-30% of the total dose ended up in blood. The exact mechanism for this translocation remains unknown at this stage. The rapidity of the process makes it unlikely that phagocytosis by macrophages and/or endocytosis by epithelial and endothelial cells are responsible for the passage. Moreover, the observation that similar proportions of particles passed into the circulation after the administration of 1, 10, or 100 µg per animal suggests a passive rather than an active, saturable phenomenon. Further work is necessary to clarify this issue. As shown in filtration experiments, before the particles are instilled, the majority is individualized; however, we do not know what happens to them in the lungs and whether they move across the lung air-blood barrier individually or not. Our results are compatible with studies that have concluded that the lung-blood barrier behaves as a molecular sieve allowing the passage of small solutes, but restricting the passage of macromolecules with the size of albumin or larger [see the review by Hermans and Bernard (29)]. Thus, Conhaim and coworkers (30) found that the lung epithelial barrier was best fitted by a three-pore-sized model, including a small number (2%) of large-sized pores (400-nm pore radius), an intermediate number (30%) of medium-sized pores (40-nm pore radius), and a very large number (68%) of small-sized pores (1.3-nm pore radius). The existence of such pores through which proteins may diffuse passively has justified the measurement of "pneumoproteins" in blood or urine to monitor damage taking place in the lungs (31). Strangely the possible passage of foreign particles through these pores does not appear to have attracted much attention to date. However, this concept is gaining acceptance in pharmacology for the administration of macromolecular drugs by inhalation (32). Moreover, the exact anatomical location of this passage remains to be established.

In conclusion, a substantial fraction of intratracheally instilled ^99mTc-albumin nanocolloid particles, taken as a model of ultrafine particles, rapidly diffuses from the lungs into the systemic circulation. This opens up the possibility that the presence of pollutant particles in the blood may lead to adverse effects on hemostasis or cardiac function.