Industrial worker exposure to airborne particles during the packing of pigment and nanoscale titanium dioxide.

CONTEXT: Titanium dioxide (TiO2) factory workers' source specific exposure and dose to airborne particles was studied extensively for particles between 5 nm and 10 µm in size. OBJECTIVE: We defined TiO2 industry workers' quantitative inhalation exposure levels during the packing of pigment TiO2 (pTiO2) and nanoscale TiO2 (nTiO2) material from concentrations measured at work area. METHODS: Particle emissions from different work events were identified by linking work activity with the measured number size distributions and mass concentrations of particles. A lung deposit model was used to calculate regional inhalation dose rates in units of particles min⁻¹ and µg min⁻¹ without use of respirators. RESULTS: Workers' average exposure varied from 225 to 700 µg m⁻³ and from 1.15 × 104 to 20.1 × 104 cm⁻4. Over 90% of the particles were smaller than 100 nm. These were mainly soot and particles formed from process chemicals. Mass concentration originated primarily from the packing of pTiO2 and nTiO2 agglomerates. The nTiO2 exposure resulted in a calculated dose rate of 3.6 × 106 min⁻¹ and 32 µg min⁻¹ where 70% of the particles and 85% of the mass was deposited in head airways. CONCLUSIONS: The recommended TiO2 exposure limits in mass by NIOSH and in particle number by IFA were not exceeded. We recommend source-specific exposure assessment in order to evaluate the workers' risks. In nTiO2 packing, mass concentration best describes the workers' exposure to nTiO2 agglomerates. Minute dose rates enable the simulation of workers' risks in different exposure scenarios.

Genotoxic effects of nanosized and fine TiO2.

The in-vitro genotoxicity of nanosized TiO(2) rutile and anatase was assessed in comparison with fine TiO(2) rutile in human bronchial epithelial BEAS 2B cells using the single-cell gel electrophoresis (comet) assay and the cytokinesis-block micronucleus test. BEAS 2B cells were exposed to eight doses (1-100 microg/cm(2)) of titanium(IV) oxide nanosized rutile (>95%, <5% amorphous SiO(2) coating; 10 x 40 nm), nanosized anatase (99.7%; <25 nm), or fine rutile (99.9%; <5 microm) for 24, 48, and 72 h. Fine rutile reduced cell viability at lower doses than nanosized anatase, which was more cytotoxic than nanosized rutile. In the comet assay, nanosized anatase and fine rutile induced DNA damage at several doses with all treatment times. Dose-dependent effects were seen after the 48- and 72-h treatments with nanosized anatase and after the 24-, 48- (in one out of two experiments), and 72-h treatments (one experiment) with fine rutile. The lowest doses inducing DNA damage were 1 microg/cm(2) for fine rutile and 10 microg/cm( 2) for nanosized anatase. Nanosized rutile showed a significant induction in DNA damage only at 80 microg/cm(2) in the 24-h treatment and at 80 and 100 microg/ cm(2) in the 72-h treatment (with a dose-dependent effect). Only nanosized anatase could elevate the frequency of micronucleated BEAS 2B cells, producing a significant increase at 10 and 60 microg/cm( 2) after the 72-h treatment (no dose-dependency). At increasing doses of all the particles, MN analysis became difficult due to the presence of TiO(2) on the microscopic slides. In conclusion, our studies in human bronchial epithelial BEAS 2B cells showed that uncoated nanosized anatase TiO(2) and fine rutile TiO(2) are more efficient than SiO( 2)-coated nanosized rutile TiO(2) in inducing DNA damage, whereas only nanosized anatase is able to slightly induce micronuclei.

Preparation of nanoparticle dispersions for in-vitro toxicity testing.

Studies on potential toxicity of engineered nanoparticle (ENP) in biological systems require a proper and accurate particle characterization to ensure the reproducibility of the results and to understand biological effects of ENP. A full characterization of ENP should include various measurements such as particle size and size distribution, shape and morphology, crystallinity, composition, surface chemistry, and surface area of ENP. It is also important to characterize the state of ENP dispersions. In this study, four different ENPs, rutile and anatase titanium dioxides and short single- and multi-walled carbon nanotubes, were characterized in two dispersion media: bronchial epithelial growth medium, used for bronchial epithelial BEAS cells, and RPMI-1640 culture media with 10% of fetal calf serum (FCS) for human mesothelial (MeT-5A) cells. The purpose of this study was to determine the characteristics of ENPs and their dispersions as well as to compare dispersion additives suitable for toxicity tests and thus establish an appropriate way to prepare dispersions that performs well with the selected ENP. Dispersion additives studied in the media were bovine serum albumin (BSA) as a protein resource, dipalmitoyl phosphatidylcholine (DPPC) as a model lung surfactant, and combination of BSA and DPPC. Dispersions were characterized using optical microscopy and transmission electron microscopy. Our results showed that protein addition, BSA or FCS, in cell culture media generated small agglomerates of primary particles with narrow size variations and improved the stability of the dispersions and thus also the relevance of the in-vitro genotoxicity tests to be done.

DNA copy number loss and allelic imbalance at 2p16 in lung cancer associated with asbestos exposure.

Five to seven percent of lung tumours are estimated to occur because of occupational asbestos exposure. Using cDNA microarrays, we have earlier detected asbestos exposure-related genomic regions in lung cancer. The region at 2p was one of those that differed most between asbestos-exposed and non-exposed patients. Now, we evaluated genomic alterations at 2p22.1-p16.1 as a possible marker for asbestos exposure. Lung tumours from 205 patients with pulmonary asbestos fibre counts from 0 to 570 million fibres per gram of dry lung, were studied by fluorescence in situ hybridisation (FISH) for DNA copy number alterations (CNA). The prevalence of loss at 2p16, shown by three different FISH probes, was significantly increased in lung tumours of asbestos-exposed patients compared with non-exposed (P=0.05). In addition, a low copy number loss at 2p16 associated significantly with high-level asbestos exposure (P=0.02). Furthermore, 27 of the tumours were studied for allelic imbalances (AI) at 2p22.1-p16.1 using 14 microsatellite markers and also AI at 2p16 was related to asbestos exposure (P=0.003). Our results suggest that alterations at 2p16 combined with other markers could be useful in diagnosing asbestos-related lung cancer.

Gene expression and copy number profiling suggests the importance of allelic imbalance in 19p in asbestos-associated lung cancer.

Asbestos is a pulmonary carcinogen known to give rise to DNA and chromosomal damage, but the exact carcinogenic mechanisms are still largely unknown. In this study, gene expression arrays were performed on lung tumor samples from 14 heavily asbestos-exposed and 14 non-exposed patients matched for other characteristics. Using a two-step statistical analysis, 47 genes were revealed that could differentiate the tumors of asbestos-exposed from those of non-exposed patients. To identify asbestos-associated regions with DNA copy number and expressional changes, the gene expression data were combined with comparative genomic hybridization microarray data. As a result, a combinatory profile of DNA copy number aberrations and expressional changes significantly associated with asbestos exposure was obtained. Asbestos-related areas were detected in 2p21-p16.3, 3p21.31, 5q35.2-q35.3, 16p13.3, 19p13.3-p13.1 and 22q12.3-q13.1. The most prominent of these, 19p13, was further characterized by microsatellite analysis in 62 patients for the differences in allelic imbalance (AI) between the two groups of lung tumors. 79% of the exposed and 45% of the non-exposed patients (P=0.008) were found to be carriers of AI in their lung tumors. In the exposed group, AI in 19p was prevalent regardless of the histological tumor type. In adenocarcinomas, AI in 19p appeared to occur independently of the asbestos exposure.

Amphibole fibres in Chinese chrysotile asbestos.

Ten chrysotile bulk samples originating from six Chinese chrysotile mines were studied for amphibole fibres. Five of the mines operate on ultramafic rocks whereas one exploits a dolomite-hosted deposit. The asbestos fibre content in lung tissue was examined from seven deceased workers of the Shenyang asbestos plant using these raw materials. The bulk samples were pretreated with acid/alkali-digestion, and thereafter, scanning and transmission electron microscopy, X-ray microanalysis, selected area electron diffraction and X-ray powder diffractometry were used to identify the minerals. Sample preparation of lung tissue involved drying and low-temperature ashing. All of the bulk samples contained amphibole fibres as an impurity. The amphibole asbestos contents were between 0.002 and 0.310 w-%. Tremolite fibres were detected in every sample but anthophyllite fibres were present only in the sample originating from the dolomite-hosted deposit. In comparison, anthophyllite (71%), tremolite (9%) and chrysotile (10%) were the main fibre types in the lung tissue samples indicating faster pulmonary clearance of chrysotile fibres. The total levels ranged from 2.4 to 148.3 million fibres (over 1 microm in length) per gram of dry tissue, and they were consistent with heavy occupational exposure to asbestos.

Pulmonary mineral fibers after occupational and environmental exposure to asbestos in the Russian chrysotile industry.

BACKGROUND: As an indicator of occupational, domestic, and environmental exposure, the level and type of asbestos fibers were determined from lung tissue samples of workers and residents who resided in the area of the world's largest asbestos mine at Asbest, Russia. METHODS: Electron microscopy was used to analyze and measure the concentration of asbestos fibers in a series of 47 autopsies at the Asbest Town Hospital. Work histories were obtained from pathology reports and employment records. RESULTS: In 24 chrysotile miners, millers, and product manufacturers, the pulmonary concentrations of retained fibers (over 1 microm in length) were 0. 8-50.6 million f/g for chrysotile, and < 0.1-1.9 million f/g for amphiboles (tremolite and anthophyllite). The concentrations were lower in 23 persons without any known occupational contact with asbestos; 0.1-14.6 million f/g for chrysotile, and < 0.1-0.7 million f/g for amphiboles. On average, 90% of all inorganic fibers were chrysotile, and 5% tremolite/anthophyllite. No amosite or crocidolite fibers were detected in any of the samples. CONCLUSIONS: The mean and range of pulmonary chrysotile concentrations were about the same as reported previously from the Canadian mining and milling industry. In the Russian samples, the mean concentration of tremolite fibers were less by at least one order of magnitude. Occupational contact was the most important source of asbestos exposure.

Asbestos bodies in bronchoalveolar lavage in relation to asbestos bodies and asbestos fibres in lung parenchyma.

In Finland, unlike other countries, anthophyllite asbestos has been widely used due to its domestic production in 1918-1975. In this particular context, the aim of the present study was to analyse the relationship between asbestos bodies (ABs) in bronchoalveolar lavage (BAL) fluid and the concentration of ABs and the different amphibole asbestos fibres in lung tissue. Sixty five BAL lung tissue sample pairs from patients with pulmonary disease were analysed. The concentration of ABs in BAL fluid and lung tissue was determined with optical microscopy, and the concentration, type and dimensions of asbestos fibres in lung tissue with scanning electron microscopy. There was a significant correlation between the concentrations of ABs in BAL fluid and in lung tissue (r = 0.72; p < 0.001), between the concentrations of ABs and amphibole asbestos fibres in lung tissue (r = 0.73; p < 0.001), and between the concentration of ABs in BAL fluid and the concentration of amphibole asbestos fibres in lung tissue (r = 0.64; p < 0.001). In patients who had been exposed mainly to commercial anthophyllite, significantly higher concentrations of ABs were observed per total pulmonary amphibole fibre burden, as compared to patients whose main exposure was to crocidolite/amosite. The anthophyllite fibres in lung tissue were longer than the crocidolite/amosite fibres. The relationship between asbestos body counts in lung tissue and in bronchoalveolar lavage fluid was similar to previous international observations. When using the asbestos body count to predict the underlying total pulmonary amphibole asbestos burden in Finnish patients, however, it should be borne in mind that the relationship between the two parameters seems to be different with anthophyllite as compared to crocidolite/amosite fibres.

Pulmonary asbestos bodies and asbestos fibers as indicators of exposure.

OBJECTIVES: The aim of the study was to analyze the correlation between pulmonary concentrations of asbestos bodies and asbestos fibers and to characterize asbestos body counts from lung tissue of Finnish patients occupationally exposed and unexposed to asbestos. METHODS: Ninety-nine surgically treated lung cancer patients were investigated. The number of asbestos bodies in iron-stained 5-micrometers histological lung tissue sections was determined by optical microscopy, and the pulmonary concentration of asbestos fibers was assessed by scanning electron microscopy. The correlation between asbestos body and asbestos fiber counts was calculated with linear regression. The asbestos body and asbestos fiber concentrations were also compared with exposure history according to a personal interview of the patients. RESULTS: The average number of asbestos bodies ranged from < 0.1 to 750 asbestos bodies per tissue section. All the cases with definite exposure showed an average of at least one asbestos body per tissue section. An average of at least one asbestos body per section was, however, detected in 34% of the patients with unlikely exposure. The regression equation log (AF) = -0.429 + 0.600.log (AB) was found to predict the concentration of asbestos fibers (AF, 10(6) fibers.g-1) corresponding to a given number of asbestos bodies (AB) in a section of lung tissue. CONCLUSIONS: The background level of asbestos bodies in the lungs of patients with no specific asbestos exposure seems to be higher in Finland than in other countries. In medicolegal cases, the methodological variation involved in asbestos fiber and asbestos body counting must be recognized and all available exposure data should be used to produce the best possible estimate of the exposure.

Asbestos exposure and the risk of lung cancer in a general urban population.

OBJECTIVES: The aim of the study was to investigate the asbestos-associated risk of lung cancer according to histological type of cancer, lobe of origin, pulmonary concentration, and type of amphibole fibers and also to estimate the etiologic fraction of asbestos for lung cancer. METHODS: The pulmonary concentration of asbestos fibers in 113 surgically treated male lung cancer patients and 297 autopsy cases among men serving as referents was determined by scanning electron microscopy. The age- and smoking-adjusted odds ratios of lung cancer were calculated according to pulmonary fiber concentration for all lung cancer types, squamous-cell carcinoma, and adenocarcinoma and for the lower-lobe and the upper- and middle-lobe cancers. RESULTS: The risk of lung cancer was increased according to the pulmonary concentration of asbestos fibers (f) of 1.0 to 4.99 x 10(6) f.g-1 [odds ratio (OR) 1.7] and > or = 5.0 x 10(6) f.g-1 (OR 5.3). The odds ratios associated with fiber concentrations of > or = 1.0 x 10(6) f.g-1 were higher for adenocarcinoma (OR 4.0) than for squamous-cell carcinoma (OR 1.6). The asbestos-associated risk was higher for lower lobe tumors than for upper lobe tumors. The risk estimates for anthophyllite and crocidolite-amosite fibers were similar, except for the risk of squamous-cell carcinoma. An etiologic fraction of 19% was calculated for asbestos among male surgical lung cancer patients in the greater Helsinki area. CONCLUSIONS: Past exposure to asbestos is a significant factor in the etiology of lung cancer in southern Finland. The asbestos-associated risk seems to be higher for pulmonary adenocarcinoma and lower-lobe tumors than for squamous-cell carcinoma and upper-lobe tumors.

Pleural plaques and exposure to mineral fibres in a male urban necropsy population.

OBJECTIVES: The study aimed to evaluate the risk of pleural plaques according to the degree of past exposure to asbestos, type of amphibole asbestos, and smoking, as well as to estimate the aetiologic fraction of asbestos as a cause of plaques among urban men. METHODS: The occurrence and extent of pleural plaques were recorded at necropsies of 288 urban men aged 33 to 69 years. The pulmonary concentration of asbestos and other mineral fibres was analysed with scanning electron microscopy. The probability of past exposure was estimated from the last occupation. RESULTS: Pleural plaques were detected in 58% of the cases and their frequency increased with age, probability of past occupational exposure to asbestos, pulmonary concentration of asbestos fibres, and smoking. The risk of both moderate and widespread plaques was raised among asbestos exposed cases, and the risk estimates were higher for widespread plaques than for moderate plaques. The age adjusted risk was higher for high concentrations of crocidolite/amosite fibres than for anthophyllite fibres. The aetiologic fraction of pulmonary concentration of asbestos fibres exceeding 0.1 million fibres/g was 43% for widespread plaques and 24% for all plaques. The median pulmonary concentrations of asbestos fibres were about threefold greater among cases with widespread plaques than among those without plaques. No increased risk of pleural plaques was associated with raised total concentrations of non-asbestos fibres. CONCLUSION: The occurrence of pleural plaques correlated closely with past exposure to asbestos. The risk was dependent on the intensity of exposure. Due to methodological difficulties in detecting past exposures to chrysotile and such low exposures that may still pose a risk of plaques, the aetiologic fractions calculated in the study probably underestimate the role of asbestos.

Asbestos exposure and pulmonary fiber concentrations of 300 Finnish urban men.

OBJECTIVES: The aim of the study was to determine the pulmonary concentrations of mineral fibers in the Finnish male urban population and to evaluate the analysis of pulmonary fiber burden by scanning electron microscopy (SEM) as an indicator of past fiber exposure. METHODS: The pulmonary concentration of mineral fibers was determined by SEM and compared with occupational history for a series of 300 autopsies of urban men aged 33 to 69 years. RESULTS: The concentration of fibers (f) longer than 1 micron ranged from < 0.3 to 163.10(6) per gram of dry tissue (f.g-1). Asbestos fiber concentrations exceeding 1.10(6) f.g-1 were observed in 33% of the cases with probable occupational exposure to asbestos and 1% of the cases with unlikely occupational exposure. Even asbestos fiber concentrations of 0.3 to 1.10(6) f.g-1, especially of crocidolite-amosite fibers, were rare among the men with unlikely occupational exposure. Fiber concentrations exceeding or equaling 1.10(6) f.g-1 were 10 times more frequent among the men more than 60 years of age as compared with those less than 40 years of age. Inorganic fibers other than asbestos had a weaker correlation with occupational history and age. Smoking habits had no significant effect on the pulmonary fiber counts. CONCLUSIONS: Asbestos fiber concentrations exceeding 1.10(6) f.g-1 are highly indicative of past occupational exposure to asbestos. The distribution of fiber concentrations in the different age groups of this study indicated decreasing asbestos exposure in Finland since the 1970s.

Gap junctional intercellular communication of primary and asbestos-associated malignant human mesothelial cells.

We examined gap junctional intercellular communication (GJIC) of primary human mesothelial cells and cell lines of asbestos-associated human pleural mesotheliomas, and the effect of asbestos and other mineral fibres on these cells. In homologous cultures, the GJIC capacity of six out of seven tumour cell lines was markedly less than for primary mesothelial cells. This defect in GJIC appeared not to be at the expression level of mRNA and protein of the gene encoding the 43 kDa gap junction protein. In heterologous cocultures of tumour cells and primary mesothelial cells, however, 80-90% of the tumour cell/normal cell contacts were functional. Exposure of primary mesothelial cells to TPA, a phorbol ester tumour promoter, resulted in marked inhibition of GJIC, being an action common to numerous tumour promoters. Such an effect though was not observed with the carcinogenic mesothelioma-inducing mineral fibres chrysotile and amosite, neither with glass wool. These results suggest that a permanent defect in GJIC capacity is a common feature of human mesothelioma cells, but how mineral fibres are involved in the process of mesotheliomagenesis is still unclear.

Cytotoxicity and anaphase aberrations induced by mineral fibres in cultured human mesothelial cells.

The in vitro cytotoxicity of two amphibole asbestos fibres (amosite and crocidolite), a serpentine asbestos (chrysotile), a non-asbestos fibrous aluminosilicate (erionite) and three different size fractions of both glass wool and rock wool fibres were assessed in an immortalized human mesothelial cell line, MeT-5A. We also investigated the induction of anaphase aberrations by the asbestos and erionite fibres. On a comparison by weight, amosite, crocidolite and chrysotile showed similar toxic effects (2-5 mug/cm(2) of the asbestos fibres caused 50% of cells to die) but erionite was less toxic (10-20 mug/cm(2) was needed for the same effect). When the doses were converted to the number of fibres/cm(2) of culture area, amosite was shown to be about 10 times more cytotoxic than crocidolite and chrysotile. Crocidolite and chrysotile showed similar cytotoxicity, and erionite was again less toxic. Of the man-made mineral fibres (MMMF), thin glass wool was the most cytotoxic (50% cell death for 10-20 mug/cm(2)), followed (in descending order of cytotoxicity) by thin rock wool, coarse glass wool, milled rock wool, milled glass wool and coarse rock wool. In general, the MMMF samples were less toxic than the asbestos and erionite samples. All three asbestos types studied induced anaphase aberrations at high (near toxic) doses. A statistically significant increase in the number of aberrant anaphases was observed in cultures treated with crocidolite or chrysotile at 5 mug/cm(2). The increase was caused by lagging chromatids, chromosomes or chromosome fragments.

Occupational exposure to asbestos as evaluated from work histories and analysis of lung tissues from patients with mesothelioma.

The past occupational exposure to asbestos of 23 patients with mesothelioma (21 men and two women) has been evaluated by a personal interview of their work history and by determination of the fibre burden in their lung tissue with scanning electron microscopy (SEM) and x ray microanalysis. According to the work history, nine patients (39%) had definitely been or probably been exposed to asbestos, six patients (26%) had had possible exposures, and eight patients (35%) unlikely or unknown exposure to asbestos. The two female patients were in the unknown exposure category. The fibre concentrations in the patients' lung tissue ranged from less than 0.1 million to 370 million fibres (f) per g dry tissue. Concentrations of over one million f per g dry tissue were found in 15 patients (65%). The lung fibre concentrations of all nine male office workers analysed for reference were less than one million f per g dry tissue. Seventy eight per cent of the patients with mesothelioma had at least possible exposure according to their history of work or concentrations of more than one million f per g dry tissue.

Asbestos-related malignant mesothelioma: growth, cytology, tumorigenicity and consistent chromosome findings in cell lines from five patients.

Seven mesothelioma cell lines were established from clinical specimens from five patients with asbestos-related malignant pleural mesothelioma. The cells in culture show either epithelial or mixed epithelial/fibrosarcomatous growth with an average doubling time of 30 h. Giant multinucleated cells are common in all the cell lines, as well as long thin microvilli on the cell surfaces. All cell lines were cytokeratin positive and they stained negatively for monocyte-macrophage markers. All seven cell lines and one long-term tissue culture from a sixth mesothelioma patient were characterized cytogenetically. Karyotype analyses revealed complex structural and numerical abnormalities, primarily involving chromosome 1, 4, 5, 6, 7, 8, 9, 11, 12, 13 and 22. An excess of chromosome material of the short arm of chromosome 5 was seen consistently in six cell lines and in the long-term culture. In cell lines from four patients, changes in chromosome 13, mainly monosomy 13, were observed. The marker chromosomes observed in the early passages were conserved and few additional changes appeared in later passages. Six of the cell lines tested for tumorigenicity in athymic nude mice were weakly positive.

Effect of two particle surface-modifying agents, polyvinylpyridine-N-oxide and carboxymethylcellulose, on the quartz and asbestos mineral fiber-induced production of reactive oxygen metabolites by human polymorphonuclear leukocytes.

We studied the capacity of quartz and asbestos fibers to induce the generation of reactive oxygen metabolites in human polymorphonuclear leukocytes (PMNs) with a chemiluminescence (CL) assay. On an equal weight basis, the particulates induced CL in the following order of magnitude: chrysotile, quartz greater than amosite, crocidolite, greater than anthophyllite, wollastonite. The intensity of CL correlated positively with the Alcian blue (a cationic dye) binding capacity of the particles. Polyvinylpyridine-N-oxide (0.5 microgram/ml) inhibited completely the CL induced by quartz but had little effect on the CL induced by asbestos fibers. Carboxymethylcellulose (1.0 microgram/ml), however, reduced the CL caused by chrysotile asbestos but had no effect on the CL induced by the other particulates. Our results suggest that in addition to length and diameter, the effect of quartz and asbestos fibers on inflammatory cells will depend on surface characteristics, including the charge of the particles.

Mineral fiber concentration in lung tissue of mesothelioma patients in Finland.

The mineral fibers in lung tissue samples of 19 mesothelioma patients and 15 randomly selected autopsy cases were analyzed using low-temperature ashing, scanning electron microscopy (SEM) and x-ray microanalysis. The fiber concentration ranged from 0.5 to 370 million fibers per gram of dry tissue in the mesothelioma group and from less than 0.01 to 3.2 million fibers per gram of dry tissue in the autopsy group. In 80% of the mesothelioma patients and in 20% of the autopsy cases, the fiber concentration exceeded 1 million fibers per gram of dry tissue. Amphibole asbestos fibers predominated in both groups, and only a few chrysotile fibers were found. In the lungs of six mesothelioma patients, anthophyllite was the main fiber type. The overall analytical precision of sample preparation and fiber counting with SEM was 22%.