New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals - a PARC (Partnership for the Assessment of Risk from Chemicals) project.

As a complex system governing and interconnecting numerous functions within the human body, the immune system is unsurprisingly susceptible to the impact of toxic chemicals. Toxicants can influence the immune system through a multitude of mechanisms, resulting in immunosuppression, hypersensitivity, increased risk of autoimmune diseases and cancer development. At present, the regulatory assessment of the immunotoxicity of chemicals relies heavily on rodent models and a limited number of Organisation for Economic Co-operation and Development (OECD) test guidelines, which only capture a fraction of potential toxic properties. Due to this limitation, various authorities, including the World Health Organization and the European Food Safety Authority have highlighted the need for the development of novel approaches without the use of animals for immunotoxicity testing of chemicals. In this paper, we present a concise overview of ongoing efforts dedicated to developing and standardizing methodologies for a comprehensive characterization of the immunotoxic effects of chemicals, which are performed under the EU-funded Partnership for the Assessment of Risk from Chemicals (PARC).

Hazard characterization of Alternaria toxins to identify data gaps and improve risk assessment for human health.

Fungi of the genus Alternaria are ubiquitous plant pathogens and saprophytes which are able to grow under varying temperature and moisture conditions as well as on a large range of substrates. A spectrum of structurally diverse secondary metabolites with toxic potential has been identified, but occurrence and relative proportion of the different metabolites in complex mixtures depend on strain, substrate, and growth conditions. This review compiles the available knowledge on hazard identification and characterization of Alternaria toxins. Alternariol (AOH), its monomethylether AME and the perylene quinones altertoxin I (ATX-I), ATX-II, ATX-III, alterperylenol (ALP), and stemphyltoxin III (STTX-III) showed in vitro genotoxic and mutagenic properties. Of all identified Alternaria toxins, the epoxide-bearing analogs ATX-II, ATX-III, and STTX-III show the highest cytotoxic, genotoxic, and mutagenic potential in vitro. Under hormone-sensitive conditions, AOH and AME act as moderate xenoestrogens, but in silico modeling predicts further Alternaria toxins as potential estrogenic factors. Recent studies indicate also an immunosuppressive role of AOH and ATX-II; however, no data are available for the majority of Alternaria toxins. Overall, hazard characterization of Alternaria toxins focused, so far, primarily on the commercially available dibenzo-α-pyrones AOH and AME and tenuazonic acid (TeA). Limited data sets are available for altersetin (ALS), altenuene (ALT), and tentoxin (TEN). The occurrence and toxicological relevance of perylene quinone-based Alternaria toxins still remain to be fully elucidated. We identified data gaps on hazard identification and characterization crucial to improve risk assessment of Alternaria mycotoxins for consumers and occupationally exposed workers.

Occupational exposure to inhalable pathogenic microorganisms in waste sorting.

This study assessed microorganisms in personal inhalable work air samples aiming to identify potential human pathogens, and correlate exposure to adverse health outcomes in waste workers. Full-shift personal exposure was measured in six different waste sorting plants. Microbial concentrations in inhalable air samples were analysed using MALDI-TOF MS for cultivable, and next generation sequencing (NGS) for non-cultivable microorganisms. Concentrations of bacterial and fungal CFUs varied substantially within and between waste sorting plants, ranging from no identifiable organisms to a maximum concentration in the order of 105 CFU/m3. Bacillus and Staphylococcus were among the most abundant bacterial genera, whilst fungal genera were dominated by Aspergillus and Penicillium. Approximately 15% of all identified species were human pathogens classified in risk group 2, whereas 7% belonged to risk group 1. Furthermore, significant correlations between concentrations of fungi in risk group 1 and self-reported adverse symptoms, such as wheezing were identified in exposed workers. The combination of culture-based methods and NGS facilitated the investigation of infectious microbial species with potential pathophysiological properties as well as non-infectious biological agents in inhalable work air samples and thereby contributed to the risk assessment of occupational exposure in waste sorting.

Bioaerosol-induced in vitro activation of toll-like receptors and inflammatory biomarker expression in waste workers.

PURPOSE: Occupational exposure to bioaerosols during waste handling remains a health concern for exposed workers. However, exposure-related health effects and underlying immunological mechanisms are still poorly described. METHODS: The present study assessed the inflammatory potential of work-air samples (n = 56) in vitro and investigated biomarker expression in exposed workers (n = 69) compared to unexposed controls (n = 25). These quantitative results were compared to self-reported health conditions. RESULTS: Personal air samples provoked an activation of TLR2 and TLR4 HEK reporter cells in one-third of all samples, indicating that the work environment contained ligands capable of inducing an immune response in vitro. Monocyte levels, as well as plasma biomarker levels, such as IL-1Ra, IL-18 and TNFα were significantly higher in exposed workers, compared to the control group when confounding factors such as BMI, sex, age and smoking habits were accounted for. Furthermore, a significant exposure-related increase in midweek IL-8 levels was measured among exposed workers. Tendencies of increased prevalence of health effects of the respiratory tract were identified in exposed workers. CONCLUSION: Inhalable dust provoked TLR activation in vitro, indicating that an exposure-related immune response may be expected in susceptible workers. However, despite significant differences in inflammatory plasma biomarker levels between exposed and unexposed workers, prevalence of self-reported health effects did not differ between the groups. This may be due to the healthy worker effect, or other factors such as adequate use of personal protective respiratory devices or adaptation to the work environment with reduced activation of the immune system.

Divergent TLR2 and TLR4 Activation by Fungal Spores and Species Diversity in Dust from Waste Sorting Plants.

This manuscript presents the results of an exploratory study on the relationships between NF-κB response through Toll-like receptor (TLR) activation by dust characterized by fungal spore concentrations and species diversity. Personal total dust samples were collected from Norwegian waste sorting plants and then characterized for fungal spores and fungal species diversity, as well as for other bioaerosol components, including endotoxins and actinobacteria. The ability of the dust to induce an NF-κB response by activating TLR2 and TLR4 in vitro was evaluated, as well as the relationship between such responses and quantifiable bioaerosol components. The average concentrations of bioaerosols were 7.23 mg total dust m-3, 4.49 × 105 fungal spores m-3, 814 endotoxin units m-3, and 0.6 × 105 actinobacteria m-3. The mean diversity measurements were 326, 0.59, and 3.39 for fungal richness, evenness, and Shannon index, respectively. Overall, fungal operational taxonomic units (OTUs) belonging to the Ascomycota phylum were most abundant (55%), followed by Basidiomycota (33%) and Mucoromycota (3%). All samples induced significant NF-κB responses through TLR2 and TLR4 activation. While fungal spore levels were positively associated with TLR2 and TLR4 activation, there was a trend that fungal species richness was negatively associated with the activation of these receptors. This observation supports the existence of divergent immunological response relationships between TLR activation and fungal spore levels on one hand and between TLR activation and fungal species diversity on the other. Such relationships seem to be described for the first time for dust from waste facilities. IMPORTANCE This manuscript presents results on multifactorial characterization of bioaerosol exposure in Norwegian waste sorting plants and the potential of such airborne dust to induce NF-κB reactions through TLR2 and TLR4 activations in an in vitro reporter cell model system. Our data revealed that increasing fungal spore levels in the dust is associated with increased activation of TLR2 and TLR4, whereas increasing fungal OTU richness is associated with decreasing activation of these receptors. The NF-κB-induced responses by the collected dust represent, therefore, effective measures of potential key immunological effects induced by a complex mixture of hazardous components, including characterized factors such as endotoxins, fungal spores, bacteria, and many other uncharacterized components. The key immunological events reported here are suggested as holistic alternatives to today's bioaerosol exposure characterization approaches for epidemiological studies in the future.

An assessment of occupational exposure to bioaerosols in automated versus manual waste sorting plants.

Occupational exposure during waste sorting is associated with several health outcomes. This study obtained knowledge about the impact of work in fully automated waste sorting plants (AWSP; n = 3) vs manual waste sorting plants (MWSP; n = 3) on personal exposure (n = 71) to bioaerosols and exposure-related health effects. Personal full-shift air samples were collected using various filter-based active sampling devices that were placed in the workers' breathing zone. Personal exposure to inhalable and thoracic dust, endotoxin and microorganisms varied considerably between and within types of waste sorting plants (WSP). Workers at AWSP were on average exposed to 0.34 mg/m3 inhalable dust, 0.15 mg/m3 thoracic dust, and 51 EU/m3 endotoxins (geometric mean (GM) levels), whereas GM exposure levels at MWSP were 0.66 mg/m3 for inhalable dust, 0.44 mg/m3 for thoracic dust, and 32 EU/m3 for endotoxins. Exposure to submicronic fungal fragments did not differ between types of plants and ranged from levels below the detection limit (limit of detection, LOD) to levels in the order of 106 fragments/m3. Higher levels of fungal fragments and fungal spores were found at AWSP compared to MWSP with a GM of 2.1 × 105 spores/m3and with a GM of 1.2 × 105 spores/m3, respectively. Actinobacterial spores were found in samples from AWSP only, with exposure levels ranging from 1.9 × 104 to 1.1 × 107 spores/m3. Exposure to microbial DNA varied within and between WSP and was on average in the order of 104 copies/m3 for fungi and 105 copies/m3 for bacteria. Health symptoms, such as sneezing, congested nose and runny nose were significantly more common among exposed workers compared to the unexposed control group.

Comprehensive assessment of occupational exposure to microbial contamination in waste sorting facilities from Norway.

Introduction: It is of upmost importance to contribute to fill the knowledge gap concerning the characterization of the occupational exposure to microbial agents in the waste sorting setting (automated and manual sorting). Methods: This study intends to apply a comprehensive field sampling and laboratory protocol (culture based-methods and molecular tools), assess fungal azole resistance, as well as to elucidate on potential exposure related health effects (cytotoxicity analyses). Skin-biota samples (eSwabs) were performed on workers and controls to identify other exposure routes. Results: In personal filter samples the guidelines in one automated industry surpassed the guidelines for fungi. Seasonal influence on viable microbial contamination including fungi with reduced susceptibility to the tested azoles was observed, besides the observed reduced susceptibility of pathogens of critical priority (Mucorales and Fusarium sp.). Aspergillus sections with potential toxigenic effect and with clinical relevance were also detected in all the sampling methods. Discussion: The results regarding skin-biota in both controls´ and workers´ hands claim attention for the possible exposure due to hand to face/mouth contact. This study allowed concluding that working in automated and manual waste sorting plants imply high exposure to microbial agents.

Bioaerosol Exposure and in vitro Activation of Toll-like Receptors in a Norwegian Waste Sorting Plant.

Background: The global shift toward greener societies demands new technologies and work operations in the waste-management sector. However, progressive industrial methods do not necessarily consider workers' health. This study characterized workers' exposure to bioaerosols and investigated the bioaerosols' potential to engage the immune system in vitro. Methods: Full shift personal aerosol sampling was conducted over three consecutive days. Dust load was analyzed by gravimetry, fungal and actinobacterial spores were analyzed by scanning electron microscopy, and endotoxin by limulus amebocyte lysate (LAL) assay. In vitro exposure of HEK cells to airborne dust samples was used to investigate the potential of inducing an inflammatory reaction. Results: The total dust exposure level exceeded the recommended occupational exposure limit (OEL) of 5.0 mg/m3 in 3 out of 15 samples. The inhalable endotoxin level exceeded the recommended exposure level by a 7-fold, whereas the fungal spore level exceeded the recommended exposure level by an 11-fold. Actinobacterial spores were identified in 8 out of 14 samples. In vitro experiments revealed significant TLR2 activation in 9 out of 14 samples vs. significant TLR4 activation in all samples. Conclusion: The present study showed that the dust samples contained potentially health-impairing endotoxin, fungi, and actinobacterial levels. Furthermore, the sampled dust contained microbial components capable of inducing TLR activation and thus have the potential to evoke an inflammatory response in exposed individuals.

Algorithm to assess the presence of Aspergillus fumigatus resistant strains: The case of Norwegian sawmills.

Association between selection pressure caused by the use of azole fungicides in sawmills and the development of fungal resistance has been described. The aim of this study was to implement an algorithm to assess the presence of Aspergillus section Fumigati resistant strains in sawmills.Eighty-six full-shift inhalable dust samples were collected from eleven industrial sawmills in Norway. Different culture media were used and molecular identification to species level in Aspergillus section Fumigati was done by calmodulin sequencing and TR34/L98H and TR46/Y121F/T289A mutations were screened by real-time PCR assay and confirmed by cyp51A sequencing. Six Fumigati isolates were identified as A. fumigatus sensu stricto and two of these grew on azole-supplemented media and were further analyzed by real-time PCR. One was confirmed to be a TR34/L98H mutant.The obtained results reinforce the need to assess the presence of A. fumigatus sensu stricto resistant isolates at other workplaces with fungicide pressure.

The airborne mycobiome and associations with mycotoxins and inflammatory markers in the Norwegian grain industry.

Grain dust exposure is associated with respiratory symptoms among grain industry workers. However, the fungal assemblage that contribute to airborne grain dust has been poorly studied. We characterized the airborne fungal diversity at industrial grain- and animal feed mills, and identified differences in diversity, taxonomic compositions and community structural patterns between seasons and climatic zones. The fungal communities displayed strong variation between seasons and climatic zones, with 46% and 21% of OTUs shared between different seasons and climatic zones, respectively. The highest species richness was observed in the humid continental climate of the southeastern Norway, followed by the continental subarctic climate of the eastern inland with dryer, short summers and snowy winters, and the central coastal Norway with short growth season and lower temperature. The richness did not vary between seasons. The fungal diversity correlated with some specific mycotoxins in settled dust and with fibrinogen in the blood of exposed workers, but not with the personal exposure measurements of dust, glucans or spore counts. The study contributes to a better understanding of fungal exposures in the grain and animal feed industry. The differences in diversity suggest that the potential health effects of fungal inhalation may also be different.

Circulating miRNAs as molecular markers of occupational grain dust exposure.

Dust from grain and feed production may cause adverse health effects in exposed workers. In this study we explored circulating miRNAs as potential biomarkers of occupational grain dust exposure. Twenty-two serum miRNAs were analyzed in 44 grain dust exposed workers and 22 controls. Exposed workers had significantly upregulated miR-18a-5p, miR-124-3p and miR-574-3p, and downregulated miR-19b-3p and miR-146a-5p, compared to controls. Putative target genes for the differentially expressed miRNAs were involved in a range of Kyoto Encyclopedia of Genes and Genomes signaling pathways, and 'Pathways in cancer' and 'Wnt signaling pathway' were common for all the five miRNAs. MiRNA-diseases association analysis showed a link between the five identified miRNAs and several lung diseases terms. A positive correlation between miR-124-3p, miR-18a-5p, and miR-574-3p and IL-6 protein level was shown, while miR-19b-3p was inversely correlated with CC-16 and sCD40L protein levels. Receiver-operating characteristic analysis of the five miRNA showed that three miRNAs (miR-574-3p, miR-124-3p and miR-18a-5p) could distinguish the grain dust exposed group from the control group, with miR-574-3p as the strongest predictor of grain dust exposure. In conclusion, this study identified five signature miRNAs as potential novel biomarkers of grain dust exposure that may have potential as early disease markers.

Exposure Determinants of Wood Dust, Microbial Components, Resin Acids and Terpenes in the Saw- and Planer Mill Industry.

OBJECTIVES: Sawmill workers have an increased risk of adverse respiratory outcomes, but knowledge about exposure-response relationships is incomplete. The objective of this study was to assess exposure determinants of dust, microbial components, resin acids, and terpenes in sawmills processing pine and spruce, to guide the development of department and task-based exposure prediction models. METHODS: 2474 full-shift repeated personal airborne measurements of dust, resin acids, fungal spores and fragments, endotoxins, mono-, and sesquiterpenes were conducted in 10 departments of 11 saw- and planer mills in Norway in 2013-2016. Department and task-based exposure determinants were identified and geometric mean ratios (GMRs) estimated using mixed model regression. The effects of season and wood type were also studied. RESULTS: The exposure ratio of individual components was similar in many of the departments. Nonetheless, the highest microbial and monoterpene exposure (expressed per hour) were estimated in the green part of the sawmills: endotoxins [GMR (95% confidence interval) 1.2 (1.0-1.3)], fungal spores [1.1 (1.0-1.2)], and monoterpenes [1.3 (1.1-1.4)]. The highest resin acid GMR was estimated in the dry part of the sawmills [1.4 (1.2-1.5)]. Season and wood type had a large effect on the estimated exposure. In particular, summer and spruce were strong determinants of increased exposure to endotoxin (GMRs [4.6 (3.5-6.2)] and [2.0 (1.4-3.0)], respectively) and fungal spores (GMRs [2.2 (1.7-2.8)] and [1.5 (1.0-2.1)], respectively). Pine was a strong determinant for increased exposure to both resin acid and monoterpenes. Work as a boilerman was associated with moderate to relatively high exposure to all components [1.0-1.4 (0.8-2.0)], although the estimates were based on 13-15 samples only. Cleaning in the saw, planer, and sorting of dry timber departments was associated with high exposure estimates for several components, whereas work with transportation and stock/finished goods were associated with low exposure estimates for all components. The department-based models explained 21-61% of the total exposure variances, 0-90% of the between worker (BW) variance, and 1-36% of the within worker (WW) variances. The task-based models explained 22-62% of the total variance, 0-91% of the BW variance, and 0-33% of the WW variance. CONCLUSIONS: Exposure determinants in sawmills including department, task, season, and wood type differed for individual components, and explained a relatively large proportion of the total variances. Application of department/task-based exposure prediction models for specific exposures will therefore likely improve the assessment of exposure-response associations.

The Inhalable Mycobiome of Sawmill Workers: Exposure Characterization and Diversity.

Exposure to fungal spores has been associated with respiratory symptoms and allergic alveolitis among sawmill workers, but the complexity of sawmill workers' fungal exposure has been poorly studied. We characterized the fungal diversity in air samples from sawmill workers' breathing zones and identified differences in the richness, diversity, and taxonomic composition between companies, departments, wood types, and seasons. Full-shift personal inhalable dust samples (n = 86) collected from 11 industrial sawmill, sorting mill, and planer mill companies processing spruce and/or pine were subjected to DNA metabarcoding using the fungal internal transcribed spacer (ITS) region 2. The workers were exposed to a higher total number of operational taxonomic units (OTUs) in summer than in winter and when processing spruce than when processing pine. Workers in the saw department had the richest fungal exposure, followed by workers in the planing department and sorting of dry timber department. Sawmills explained 11% of the variation in the fungal community composition of the exposure, followed by season (5%) and department (3%). The fungal compositions of the exposures also differed between seasons, sawmills, wood types, and departments at the taxonomic level, ranging from the phylum to the species level. The differences in exposure diversity suggest that the potential health effects of fungal inhalation may also be different; hence, a risk assessment based on the fungal diversity differences should be performed. This study may serve as a basis for establishing a fungal profile of signature species that are specific for sawmills and that can be measured quantitatively in future risk assessments of sawmill workers.IMPORTANCE To gain more knowledge about exposure-response relationships, it is important to improve exposure characterization by comprehensively identifying the temporal and spatial fungal composition and diversity of inhalable dust at workplaces. The variation in the diverse fungal communities to which individuals are exposed in different seasons and sawmills suggests that variations in exposure-related health effects between seasons and companies can be expected. More importantly, the distinct fungal profiles between departments across companies indicate that workers in different job groups are differently exposed and that health risks can be department specific. DNA metabarcoding provides insight into a broad spectrum of airborne fungi that may serve as a basis for obtaining important knowledge about the fungi to which workers are exposed.

Fungal aerosol composition in moldy basements.

Experimental aerosolization studies revealed that fungal fragments including small fragments in the submicrometer size are released from fungal cultures and have been suggested to represent an important fraction of overall fungal aerosols in indoor environments. However, their prevalence indoors and outdoors remains poorly characterized. Moldy basements were investigated for airborne fungal particles including spores, submicron fragments, and larger fragments. Particles were collected onto poly-L-lysine-coated polycarbonate filters and qualitatively and quantitatively analyzed using immunogold labeling combined with field emission scanning electron microscopy. We found that the total fungal aerosol levels including spores, submicrometer, and larger fragments in the moldy basements (median: 80 × 103  m-3 ) were not different from that estimated in control basements (63 × 103  m-3 ) and outdoor (90 × 103  m-3 ). However, mixed effect modeling of the fungal aerosol composition revealed that the fraction of fragments increased significantly in moldy basements, versus the spore fraction that increased significantly in outdoor air. These findings provide new insight on the compositional variation of mixed fungal aerosols in indoor as compared to outdoor air. Our results also suggest that further studies, aiming to investigate the role of fungal aerosols in the fungal exposure-disease relationships, should consider the mixed composition of various types of fungal particles.

Predictors for Increased and Reduced Rat and Mouse Allergen Exposure in Laboratory Animal Facilities.

Introduction: Exposure to rat and mouse allergens during work in laboratory animal facilities represents a risk for being sensitized and developing allergic diseases, and it is important to keep the exposure level as low as possible. The objective of this study was to characterize the personal Mus m 1 and Rat n 1 exposure during work in laboratory animal facilities, and to investigate the effect of identified predictors of increased and reduced exposure. Methods: Mus m 1 and Rat n 1 were analysed in whole day or task-based personal air samples by enhanced sensitivity sandwich enzyme-linked immunosorbent assay. Information about cage-and-rack systems, tasks, and other conditions known to influence the allergen exposure was registered. Predictors for allergen exposure were identified by multiple linear regression analyses. Results: The median allergen exposure was 3.0 ng m-3 Mus m 1 and 0.5 ng m-3 Rat n 1, with large task-dependent variations among the samples. The highest exposed job group were animal technicians. Cage emptying and cage washing in the cage washroom represented the highest exposure, whereas animal experiments in the lab/operation room represented the lowest exposure, with laminar airflow bench being an exposure-reducing determinant. Cage changing was the highest exposed task in the animal room, where individually ventilated cages (IVCs) were predictors of reduced exposure for both Mus m 1 and Rat n 1, whereas cage-rack systems with open shelves and sliding doors were predictors of increased Rat n 1 exposure. Cages of IVC type with positive air pressure (IVC+) as well as open shelves and sliding doors were strong predictors of increased exposure during cage emptying and cage washing. Conclusions: Significant different exposure levels depending on type of work and task imply different risks of sensitization and allergy development. The fact that IVC+ cages have opposite impact on Mus m 1 and Rat n 1 exposure during different tasks may have positive clinical implications when taken into account.

Exposure to Wood Dust, Microbial Components, and Terpenes in the Norwegian Sawmill Industry.

Sawmill workers are exposed to wood dust (a well-known carcinogen), microorganisms, endotoxins, resin acids (diterpenes), and vapours containing terpenes, which may cause skin irritation, allergy, and respiratory symptoms including asthma. The health effects of most of these exposures are poorly understood as most studies measure only wood dust. The present study assessed these exposures in the Norwegian sawmill industry, which processes predominantly spruce and pine. Personal exposures of wood dust, resin acids, endotoxin, fungal spores and fragments, mono-, and sesquiterpenes were measured in 10 departments in 11 saw and planer mills. The geometric mean (GM) and geometric standard deviation (GSD) thoracic exposures were: 0.09 mg m-3 dust (GSD 2.6), 3.0 endotoxin units (EU) m-3 (GSD 4.9), 0.4 × 105 fungal spores m-3 (GSD 4.2), 2 × 105 fungal fragments m-3 (GSD 3.2), and 1560 ng m-3 of resin acids (GSD 5.5). The GM (GSD) inhalable exposures were: 0.72 mg m-3 dust (2.6), 17 EU m-3 (4.3), 0.4 × 105 fungal spores m-3 (3.8), and 7508 ng m-3 (4.4) of resin acids. The overall correlation between the thoracic and inhalable exposure was strong for resin acid (rp = 0.84), but moderate for all other components (rp = 0.34-0.64). The GM (GSD) exposure to monoterpenes and sesquiterpenes were 1105 µg m-3 (7.8) and 40 µg m-3 (3.9), respectively. Although mean exposures were relatively low, the variance was large, with exposures regularly exceeding the recommended occupational exposure limits. The exposures to spores and endotoxins were relatively high in the dry timber departments, but exposures to microbial components and mono-and sesquiterpenes were generally highest in areas where green (undried) timber was handled. Dust and resin acid exposure were highest in the dry areas of the sawmills. Low to moderate correlation between components (rp ranging from 0.02 to 0.65) suggests that investigations of exposure-response associations for these components (both individually and combined) are feasible in future epidemiological studies.

Pneumoproteins and markers of inflammation and platelet activation in the blood of grain dust exposed workers.

PURPOSE: To investigate if blood biomarkers could indicate early signs of lung damage or cardiovascular risk due to exposure to grain dust. MATERIALS AND METHODS: Pneumoproteins and markers of inflammation and platelet activation were analysed in blood samples of 102 grain elevator and compound feed mill workers. Differences between exposed (n = 67) and controls (n = 35), and associations with exposure measurements and respiratory health were investigated by multiple linear regression analyses. RESULTS: Concentrations of CC-16 and IL-6 were higher in exposed workers compared with controls (p < 0.001 for both), whereas fibrinogen was lower (p = 0.005). Concentrations of CRP, TNF-α, sCD40L and sP-selectin were similar in both groups. Serum CC-16 was significantly higher in workers with farm childhood, regardless of exposure. The impact of farm childhood on CC-16 interacted with smoking. None of the biomarkers were associated with exposure measurements or any of the tested respiratory health parameters. CONCLUSION: Dust exposure induced inflammatory and anti-inflammatory reactions, but did not induce systemic inflammation and had no effect on platelet activation. No cause-effect relationship could be established in spite of relatively high exposure levels, particularly to endotoxin. Whether increased serum CC-16 is an early sign of lung damage or a reversible defense reaction remains unclear.

Fungal Fragments and Fungal Aerosol Composition in Sawmills.

Assessment of exposure to fungi has commonly been limited to fungal spore measurements that have shown associations between fungi and development or exacerbation of different airway diseases. Because large numbers of submicronic fragments can be aerosolized from fungal cultures under laboratory conditions, it has been suggested that fungal exposure is more complex and higher than that commonly revealed by spore measurements. However, the assessment of fungal fragments in complex environmental matrix remain limited due to methodological challenges. With a recently developed immunolabeling method for field emission scanning electron microscope (FESEM), we could assess the complex composition of fungal aerosols present in personal thoracic samples collected from two Norwegian sawmills. We found that large fungal fragments (length >1 µm) dominated the fungal aerosols indicating that the traditional monitoring approach of spores severely underestimate fungal exposure. The composition of fungal aerosols comprised in average 9% submicronic fragments, 62% large fragments, and 29% spores. The average concentrations of large and submicronic fragments (0.2-1 µm) were 3 × 105 and 0.6 × 105 particles m-3, respectively, and correlated weakly with spores (1.4 × 105 particles m-3). The levels of fragments were 2.6 times higher than the average spore concentration that was close to the proposed hazardous level of 105 spores per m3. The season influenced significantly the fungal aerosol concentrations but not the composition. Furthermore, the ratio of spores in the heterogeneous fungal aerosol composition was significantly higher in saw departments as compared to sorting of green timber departments where the fungal fragments were most prevalent. Being the dominating particles of fungal aerosols in sawmills, fungal fragments should be included in exposure-response studies to elucidate their importance for health impairments. Likewise, the use of fungal aerosol composition in such studies should be considered.

Cross-shift study of exposure-response relationships between bioaerosol exposure and respiratory effects in the Norwegian grain and animal feed production industry.

OBJECTIVE: We have studied cross-shift respiratory responses of several individual bioaerosol components of the dust in the grain and feed industry in Norway. METHODS: Cross-shift changes in lung function and nasal congestion, as well as in respiratory and systemic symptoms of 56 exposed workers and 36 referents, were recorded on the same day as full-shift exposure to the inhalable aerosol fraction was assessed. Exposure-response associations were investigated by regression analysis. RESULTS: The workers were exposed on average to 1.0 mg/m(3) of grain dust, 440 EU/m(3) of endotoxin, 6 µg/m(3) of ß-1,3-glucans, 17×10(4)/m(3) of bacteria and 4×10(4)/m(3) of fungal spores during work. The exposure was associated with higher prevalence of self-reported eye and airway symptoms, which were related to the individual microbial components in a complex manner. Fatigue and nose symptoms were strongest associated with fungal spores, cough with or without phlegm was associated with grain dust and fungal spores equally strong and wheeze/tight chest/dyspnoea was strongest associated with grain dust. Bioaerosol exposure did not lead to cross-shift lung function decline, but several microbial components had influence on nose congestion. CONCLUSIONS: Exposure to fungal spores and dust showed stronger associations with respiratory symptoms and fatigue than endotoxin exposure. The associations with dust suggest that there are other components in dust than the ones studied that induce these effects.

Profiling of 3-hydroxy fatty acids as environmental markers of endotoxin using liquid chromatography coupled to tandem mass spectrometry.

3-Hydroxy acids are constituents of the lipid A part of lipopolysaccharides and may potentially be used as chemical markers of endotoxin. While commercial enzymatic assays, such as the widely used Limulus amebocyte lysate (LAL) assay, commonly detect merely the water-soluble fraction of the bioactive endotoxin, the chemical approach aims to estimate the total amount of endotoxin present in a sample. Our objective was to develop a simple method for quantitative profiling of 3-hydroxy fatty acids in occupational and environmental samples based on detection with HPLC-MS/MS. We included eleven 3-hydroxy fatty acids (3-hydroxyoctanoic acid to 3-hydroxyoctadecanoic acid) in the HPLC-MS/MS based method, which involved base hydrolysis of filter samples using 1M sodium hydroxide and removal of the base as well as concentration of the fatty acids using solid-phase extraction on a functionalized polystyrene-divinylbenzene polymer. Recovery trials from spiked glass fiber filters, using threo-9,10-dihydroxyhexadecanoic acid as internal standard, gave an overall recovery of 54-86% for 3-hydroxy fatty acids of medium chain length (3-hydroxynonanoic to 3-hydroxypentadecanoic acid). 3-Hydroxyoctanoic acid and the longer chain fatty acids were more problematic yielding overall spike recoveries of 11-39%. While the 3-hydroxy fatty acid profile of pure lipopolysaccharides was dominated by 3-hydroxydecanoic, 3-hydroxydodecanoic and 3-hydroxytetradecanoic acid the aqueous phase from drilling mud contained in addition relatively high amounts of 3-hydroxyoctanoic and 3-hydroxynonanoic acid. Endotoxin activity as measured by the LAL assay was reasonably correlated (R(2)=0.54) to the sum of 3-hydroxydecanoic acid, 3-hydroxydodecanoic acid and 3-hydroxytetradecanoic acid in these samples.