a b
c d
Figure 1. SEM pictures of a) quartzite, b) fused silica, c) pegmatite quartz, d) feldspar (bar —— 5 µm).
a) b)
SYTTY
THE EFFECT OF SURFACE PROPERTIES OF MINERAL DUSTS ON THEIR ABILITY TO INDUCE INFLAMMATORY RESPONSE IN THE LUNGS
Project leader: Matti Klockars, University of Helsinki, Department
of Public Health, Box 41,
FIN-00014 University of Helsinki, Helsinki, Finland, tel. +358-9-1912
7557,
e-mail: Matti.Klockars@helsinki.fi
| PUBLICATIONS |
| TIIVISTELMÄ SUOMEKSI |
Researchers:
Mikko Holopainen, Occupational Health Institute, Box 93, 70701 Kuopio,
tel. +358-17-201249
Maija-Riitta Hirvonen, National Public Health Institute, Box 95, 70701
Kuopio, tel. +358-17- 201211
Hannu Komulainen, National Public Health Institute, Box 95, 70701 Kuopio,
tel. +358-17-201211
Kari Kuuspalo, University of Kuopio, Lab. for Atmospheric Physics and
Chem. tel. +358-40 5310502
Markku J. Lehtinen, Geological Survey of Finland, Espoo, tel. +358-20-5502116
Financing SYTTY organisation: The Academy of Finland
Funding from SYTTY / Total funding of project (€): 73391
/ 105061
Person-months of work funded by SYTTY / Total person-months of work:
22 / 29
KEY WORDS: quartz, feldspar, mica, silicates, cytokines, inflammation
EXTENDED ABSTRACT
1 Introduction
Urban air particles originate largely from combustion processes. However, silicate particles detached from road pavements and sanding materials are significant particularly during spring dust episodes. Inhaled silicate particles have various capacities to induce inflammation in the lungs. One of the pathways by which inhaled particles stimulate the recruitment and subsequent activation of inflammatory cells is through the activation of lung macrophages to release TNFalpha. Furthermore, in vitro and in vivo studies have shown a correlation between the ability of mineral particles to produce inflammation in the lung and their capacity to activate macrophage TNFalpha production. Inflammation can be involved with the mechanisms by which urban air particles increase cardiorespiratory mortality.
Feldspars, quartz, micas, amphiboles and pyroxenes are typical groups of minerals in rocks and sands used for road pavements in Finland. A respirable size silicate particle usually consist of one particular mineral, sometimes of two or three minerals.
The potential of quartz, feldspar and mica to induce inflammatory response was investigated in this study by measuring the production of proinflammatory cytokines and NO in particle-exposed macrophages in vitro. Furthermore, we investigated if the geological origin of quartz or the plate-like shape of mica particles is important for their capacity to stimulate inflammatory responses in macrophages.
2 Methods
The silicates studied in the quartz study were 3 quartz samples of different geological origin and one feldspar sample. The samples were received from three Finnish mines. Quartzite is from Kinahmi quarry, Siilinjärvi, Eastern Finland. It represents a Precambrian (ca. 2000-2200 Ma ago) weathering product, ancient “seashore quartz sand”, which was metamorphosed (recrystallised) to orthoquartzite. Fused silica is made by heating Kinahmi quartz into high temperature (ca. 2000 °C) after which it is then cooled. By this method fine-grained lumps of silica are formed. Quartz from the mineralogically fairly simple Ala-Aulis granite pegmatite, Kemiö, SW Finland is alpha quartz. The feldspar sample represents the Haapaluoma pegmatite, Peräseinäjoki,West Finland. The ca. 1800 Ma old Haapaluoma dykes are examples of complex-pegmatites. Haapaluoma is structurally and mineralogically zoned and contains gigantic K-feldspar (microcline perthite) crystals.
Particles for the tests were produced from the above mentioned bulk quartz, fused silica and feldspar samples using modified Pitt-3 acoustical aerosol generator. From the aerosol generator the generated polydisperse aerosol was led to a vertical elutriation chamber, where redundant large particles were separated from the air stream by gravitation. Further separation of the particles was done by collecting the particles according to their aerodynamic sizes from the upper end of the elutriator on filter papers by Cascade Centripeter (Bird and Tole Cascade Centripeter, BGI inc). The size range of the collected particles was verified by electron microscopy for each separate dust type.
In the mica study, Finnish phlogopite (tetraferriphlogopite) samples, received from Kemira Oy, Finland were used. Respirable size samples were prepared either by grinding with a jet mill (milled phlogopite) or using a water elutriation method (elutriated phologopite). One phlogopite sample (acid treated phlogopite) was treated with sulfuric and nitric acids to extract cations. The sample was then washed several times with water and jet milled. The particle size distribution, mean particles size and specific surface area (Table 1) of the phlogopite samples was analyzed with an optical particle size analyzer (Malvern, Master Sizer). The specific surface area of the mineral dusts was analyzed also with a method based on nitrogen sorption (Mikrometric Flow Sorb II 2300).
Alpha quartz (min-u-sil <5µm) was used as a positive control and titanium dioxide as a negative control dust in both studies. Mineralogical purity and particle morphology of the fine dust samples were analyzed at GTK (Espoo) by scanning electron microscope (Jeol JSM LV 5900) + energy dispersed spectrometer (Link Inca) from back-scattered (BSE) and secondary electron (SE) images. Proper mineral chemistry was determined with electron probe microanalyzer (Cameca Camebax SX50).
Mouse macrophage cell line RAW264.7 was used in the study. Cells were exposed to mineral particles. The dose and time-dependent viability, production of proinflammatory cytokines (IL-6, IL-8 and TNFalpha) and NO of the cells after their exposure to different particles was studied as an indication of cytotoxicity and activation of preinflammatory mechanisms. For the dose response studies the cells were exposed to doses of 10, 50 100, 200 or 500 µg/ ml of each types of mineral particles for 24 hrs. After the exposure, the cell suspension was centrifuged. NO was analyzed in an aliquot of the supernatant by the Griess reaction as the stable NO-oxidation product nitrite. The rest of supernatants were stored at -80 °C for the analyses of TNFalpha and IL-6 which was carried out with enzyme immunoassay by using commercial ELISA kits (Pharmingen, San Diego, CA). The experiments were repeated three times in duplicate.
3 Results and discussion
The SEM (SEI) pictures taken from the particles show that the particle size of quartz and feldspar samples was of the same magnitude and therefore comparable in all samples. Typical particle size was less than 5 µm but more that 1 µm (Fig1). Phase counting from the BSE-images after identifications with EDS gave the results in table 1:
Table 1. Phase counting of mineral particles studied from back-scattered images with energy dispersed spectrometer.
| Mineral | Q/S | K | P | M | K+Q | K+P | Q+P | K+Q+P |
| Quartzite quartz | 100 | |||||||
| Fused silica | 100 | |||||||
| Pegmatite quartz | 84 | 8 | 2 | 4 | 2 | |||
| Pegmatite feldspar | 7 | 54 | 7 | 2 | 27 | 3 |
A XRD run of fused silica made at GTK showed a generally rounded diffractogram of a nearly amorphous structural state with some small peaks of quartz. No cristobalite was detected with XRD. The average main mineral composition of pegmatite quartz is: 37 % microcline, 31 % quartz, 26 % plagioclase, 5 % biotite, and 1 % muscovite + chlorite.
All analysed quartz and fused silica grains showed to be practically iron free (0.00-0.01), except one quartz grain from Haapaluoma (0.33 % FeO). The FeO-content of potassium feldspar grains from mineral dust representing Haapaluoma feldspar sample was 0.06-0.12 %. The FeO-content of microcline from a reference pegmatite ore sample from Ala-Aulis was only 0.01 %.
Silica and feldspar samples induced dose-dependent cytotoxicity at 24 hrs at the concentrations of 10, 50, 100 and 200 µg/ ml. Titanium dioxide was non-toxic at doses 10-200 µg/ ml. Fused silica was the most toxic sample whereas feldspar caused minor toxicity.
Fused silica, pegmatite quartz and min-u-sil triggered dose dependent
TNFalpha-production in macrophages, whereas relatively low response was
detected by feldspar, quartzite and particularly by titanium dioxide. Fused
silica induced greater response at the doses 10, 50 and 100 µg/ml
than the other samples (Fig.2a).
The IL-6 response in macrophages had a similar profile as in case of
TNFalpha. Fused silica, min-u-sil and pegmatite quartz induced the greatest
and dose-dependent IL-6 responses. Quartzite induced a very low IL-6 production
(Fig. 2b). The production of IL-6 was small, at most only 0.9 % from the
response by LPS. Any of the mineral particle samples did not induce
production of NO in the cells at any time point whereas LPS induced a significant
time- and dose dependent NO-response in the cells.
The elutriated phlogopite particles were larger and had smaller surface area per weight unit than milled phlogopite (Table 2). The elutriated phlogopite was more regular in shape and plate-like compared with milled phlogopite. The content of cations was very low in the acid treated phlogopite. The surface area of acid treated phlogopite was almost one hundred-fold when measured with nitrogen adsorption method compared with optical analysis of the surface area (Table 2).
Table 2. Mean particles sizes (µm) and specific surface areas (m²/g) measured with optical1 and nitrogen adsorption2 methods, of milled, acid treated and elutriated phlogopite, min-u-sil and titanium dioxide.
| jet milled
phlogopite |
acid treated
phlogopite |
elutriated
phlogopite |
min-u-sil | titanium
dioxide |
|
| mean particles size (µm) | 1,28 | 2,65 | 3,98 | 1,24 | 0,42 |
| specific surface area1 (m2/g) | 7,71 | 2,78 | 2,62 | 8,9 | |
| specific surface area2 (m2/g) | 11,7 | 225,7 | 7,6 | 3,1 | 7,4 |
All phlogopite samples and min-u-sil induced production of TNFalpha in macrophages. The dose response analysis showed the following order of potency: elutriated phlogopite > min-u-sil > acid treated phlogopite > milled phlogopite. Elutriated phlogopite caused greater response compared with milled phlogopite and min-u-sil at all doses. There was only a minor IL-6 production by mineral dust exposed macrophages. Only elutriated phlogopite induced a dose-dependent IL-6 response. None of the mineral particle samples induced production of NO in the cells.
The proinflammatory effect in vitro, seemed to depend on the characteristics of the particles. It is commonly assumed, that small size particles are more harmful. However, in our in vitro study, the elutriated mica had the largest particle size, but was still the most powerful stimulator of macrophages. SEM (SEI) photographs revealed, that elutriated phlogopite particles had a mere plate-like form, typical for mica, whereas milled particles were more diverse, as many of them had been crushed during jet milling. Hence, particle size combined with plate-like form appeared to be important, but the magnitude of the specific surface area was not a major factor. Possibly, the shape of mica particles, being both thin and wide strengthens their capacity to stimulate macrophages. The effect of aspect-ratio of mineral fibers is well known. It has been shown in many studies, that long fibers stimulate macrophages more than short fibers. The aspect ratio may have a role also in case of plate-like particles.
4 Conclusions
The low capacity of feldspar and quartzite sandstone to stimulate proinflammatory cytokines may be an advantage as far as their health effects are concerned. On the other hand, the results of this study show that quartz of igneous origin may be more harmful material.
The plate-like shape of sheet phlogopite was important for their capacity
to induce the release of proinflammatory cytokines from macrophages.
a
b
c
d
Figure 1. SEM pictures of a) quartzite, b) fused silica, c) pegmatite
quartz, d) feldspar (bar —— 5 µm).
a)
b)
Figure 2. Mineral dust induced production of TNFalpha (a) and IL-6 (b)
in RAW264.7 cells at the concentrations of 10, 50, 100, 200, and 500 µg/ml
after 24 hrs exposure (Mean and SE).