To Örebro University

BACKGROUND: Cobalt is a dermal sensitizer, and keratinocytes respond to cobalt exposure by releasing proinflammatory mediators, regulating the immune response.

OBJECTIVE: To determine the effect of cobalt on the inflammasome associated cytokine- and gene expression in cultured human keratinocytes (HaCaT). Cultivation in low- or high calcium conditions model separate differentiation states of keratinocytes in the skin.

METHOD: HaCaT cells in two different states of differentiation were exposed to cobalt chloride and caspase-1 activity as well as the production of IL-1 beta, IL-18 and gene expression of IL1B, IL18, NLRP3, CASP1, and PYCARD was quantified. 

RESULTS: High cobalt chloride exposure mediated significant increase in caspase-1 activity, cytokine levels, and IL1B and NLRP3 expression with a corresponding regulatory decrease for CASP1 and PYCARD expression. No difference between high- and low calcium culturing conditions modelling differentiation states was detected.

CONCLUSIONS: Our data suggest that HaCaT cells respond with inflammmasome associated activity upon cobalt exposure in a concentration-dependent manner. These mechanisms could be of importance for the understanding of the pathophysiology behind allergic sensitization to dermal cobalt exposure.

BACKGROUND: Sensitization requires exposure to an allergen with subsequent production of a "danger "signal. In the skin, keratinocytes are the main producers of these signals.

OBJECTIVE: To compare dose- and time-effects of cobalt on the viability of and cytokine release from HaCaT cells cultured at low or high calcium.

METHOD: To model two separate states of differentiation of keratinocytes, HaCaT cells were cultured under low or high calcium conditions. HaCaT were exposed to different concentrations of cobalt chloride (10 μm to 5 mM) over time (30 minutes- 48 hours). Cell viability was measured with the Cell-Titer Blue Viability assay. Cytokine production was measured using a bead-based immunoassay and flow cytometry. Gene expression was quantified using qPCR. Data was analyzed by ANOVA and linear mixed model.

RESULTS: Viability of the cells was dose- and time-dependent. A linear mixed statistical model showed that cobalt exposure induces increase in IL-6, CXCL8 and CCL2 production over time and whereas increase of IL-6 and a decrease of CCL2 was associated with increasing cobalt chloride concentrations. When comparing the cells incubated under high and low calcium conditions, the more differentiated cells in the high concentration were found to exert a stronger response in terms of IL-6 release.

CONCLUSIONS: Our data suggest that cobalt chloride triggered an alarm system in HaCaT cells, and proinflammatory cytokines/chemokines were secreted in a dose- and time-dependent manner. When high and low calcium incubations were compared, the difference was seen only for IL-6. These findings indicate that the effect of cobalt chloride on cell toxicity occurs throughout the living epidermis.

Cobalt is a strong sensitizer and can cause contact allergy upon both direct contact or from airborne exposure on the skin. In the skin, keratinocytes are the first cells to come in contact with the metal and will react and respond to the danger by triggering an alarm system resulting in an inflammatory response in the skin. Keratinocytes have been shown to produce IL-1β, which is one of the most potent  inflammatory agents in our body and is associated with a variety of diseases.

The aims of this thesis was to investigate cobalt air concentrations for different particle fractions for possible use as proxies for other article measures and to examine if cobalt skin and inhalable air exposure contributes to uptake. Also, to investigate the effect of cobalt on cultured human keratinocyte cell viability, pro-inflammatory cytokine/chemokine release and NLRP3 inflammasome activation using cells cultured at low or high calcium (the latter yielding a more differentiated cell type).

Air exposure to cobalt was found in all departments and for all work tasks in the hard metal production facility and exposures were in general below the Swedish OEL for inhalable cobalt. The highest exposure levels were found in the powder production department and for laboratory and furnace work. Good correlations for the mass based measures enable us to use the findings for future references. When personal inhalable air levels of cobalt, cobalt skin levels skin and biological monitoring of cobalt in blood were analysed, the skin exposure was determined to be import as a route of uptake. Skin exposure to cobalt in the hard metal industry, could further affect the total uptake in the same order of magnitude as air exposure. In vitro investigations of cobalt using the human keratinocyte cell line HaCaT, showed that CoCl2 triggered an alarm system in cells where the proinflammatory cytokines/chemokines IL-6, CXCL8 and CCL2, known to be involved in skin inflammation, were secreted in a time- and dosedependent manner. Comparing HaCaT cells of high- and low differentiation stages indicated that the effect of cobalt chloride on cell toxicity occurs throughout the living epidermis. CoCl2 exposure also resulted in secretion of the proinflammatory cytokines IL-1β and IL-18, and caspase-1, which indicates activation of the NLRP3 inflammasome in the cells. CoCl2 regulates the inflammasome both as primer and as an activator. Our mRNA results indicates a negative feedback mechanism in the inflamamsome due to the exposure. The inflammatory response in general is more dose than time dependent, which be important for understanding the mechanisms of allergic sensitization.

Background: Occupational exposure to cobalt is well established in hard metal manufacture. Cobalt is known to cause contact allergy, asthma, hard metal lung disease, and lung cancer. The relationship between skin exposure and uptake determined in blood has not been extensively investigated.

Objective: To examine whether skin and inhalable air exposure to cobalt contributes to uptake, determined as cobalt in blood, in a hard metal manufacturing factory.

Methods: The amount of cobalt on the skin found with an acid wash technique, the air concentrations of inhalable cobalt and cobalt blood concentrations were determined and correlated in exposed workers.

Results: We found a significant rank correlation for cobalt concentrations on the skin, in inhalable air, and in blood (0.376-0.498). Multiple linear regression showed significant regression coefficients for cobalt skin exposure and blood (B = 0.01, p < 0.05) and for inhalable cobalt in air and blood (B = 49.1, p < 0.001). According to our model based on data from the regression analyses, a twofold increase in skin exposure levels at different air concentrations caused a 3 - 14% increase in blood levels.

Conclusions: Our data suggest that skin exposure to cobalt in the hard metal industry could affect the total uptake at the same order of magnitude as air exposure.

Exposure to cobalt in the hard metal industry entails severe adverse health effects, including lung cancer and hard metal fibrosis. The main aim of this study was to determine exposure air concentration levels of cobalt and tungsten for risk assessment and dose-response analysis in our medical investigations in a Swedish hard metal plant. We also present mass-based, particle surface area, and particle number air concentrations from stationary sampling and investigate the possibility of using these data as proxies for exposure measures in our study. Personal exposure full-shift measurements were performed for inhalable and total dust, cobalt, and tungsten, including personal real-time continuous monitoring of dust. Stationary measurements of inhalable and total dust, PM2.5, and PM10 was also performed and cobalt and tungsten levels were determined, as were air concentration of particle number and particle surface area of fine particles. The personal exposure levels of inhalable dust were consistently low (AM 0.15mg m(-3), range <0.023-3.0mg m(-3)) and below the present Swedish occupational exposure limit (OEL) of 10mg m(-3) The cobalt levels were low as well (AM 0.0030mg m(-3), range 0.000028-0.056mg m(-3)) and only 6% of the samples exceeded the Swedish OEL of 0.02mg m(-3) For continuous personal monitoring of dust exposure, the peaks ranged from 0.001 to 83mg m(-3) by work task. Stationary measurements showed lower average levels both for inhalable and total dust and cobalt. The particle number concentration of fine particles (AM 3000 p·cm(-3)) showed the highest levels at the departments of powder production, pressing and storage, and for the particle surface area concentrations (AM 7.6 µm(2)·cm(-3)) similar results were found. Correlating cobalt mass-based exposure measurements to cobalt stationary mass-based, particle area, and particle number concentrations by rank and department showed significant correlations for all measures except for particle number. Linear regression analysis of the same data showed statistically significant regression coefficients only for the mass-based aerosol measures. Similar results were seen for rank correlation in the stationary rig, and linear regression analysis implied significant correlation for mass-based and particle surface area measures. The mass-based air concentration levels of cobalt and tungsten in the hard metal plant in our study were low compared to Swedish OELs. Particle number and particle surface area concentrations were in the same order of magnitude as for other industrial settings. Regression analysis implied the use of stationary determined mass-based and particle surface area aerosol concentration as proxies for various exposure measures in our study.