Comparative cytotoxicity of respirable surface-treated/untreated calcium carbonate rock dust particles in vitro

Abstract

Calcium carbonate rock dust (RD) is used in mining to reduce the explosivity of aerosolized coal. During the dusting procedures, potential for human exposure occurs, raising health concerns. To improve RD aerosolization, several types of anti-caking surface treatments exist. The aim of the study was to evaluate cytotoxicity of four respirable RD samples: untreated/treated limestone (UL/TL), untreated/treated marble (UM/TM), and crystalline silica (SiO₂) as a positive control in A549 and THP-1 transformed human cell lines. Respirable fractions were generated and collected using FSP10 high flow-rate cyclone samplers. THP-1 cells were differentiated with phorbol-12-myristate-13-acetate (20 ng/ml, 48 h). Cells were exposed to seven different concentrations of RD and SiO₂ (0-0.2 mg/ml). RD caused a slight decrease in viability at 24 or 72 h post-exposure and were able to induce inflammatory cytokine production in A549 cells, however, with considerably less potency than SiO₂. In THP-1 cells at 24 h, there was significant dose-dependent lactate dehydrogenase, inflammatory cytokine and chemokine release. Caspase-1 activity was increased in SiO₂- and, on a lesser scale, in TM- exposed cells. To test if the increased toxicity of TM was uptake-related, THP-1 cells were pretreated with Cytochalasin D (CytD) or Bafilomycin A (BafA), followed by exposure to RD or SiO₂ for 6 h. CytD blocked the uptake and significantly decreased cytotoxicity of all particles, while BafA prevented caspase-1 activation but not cytotoxic effects of TM. Only TM was able to induce an inflammatory response in THP-1 cells, however it was much less pronounced compared to silica.

Keywords: Limestone; Macrophages; Marble; Rock dust; Silica; Stearate.

Fig. 1.

Stearate-based treatment of Calcium carbonate particulates, partially reprinted from (Cao et al., 2016), with permission from Elsevier.

Fig. 2.

Representative TEM images of respirable rock dust samples. UL - untreated limestone, TL – treated limestone, UM – untreated marble, TM – treated marble.

Fig. 3.

Viability of the A549 cells following 24 h (A) and 72 h (B) exposure to various rock dusts and silica. Values are expressed as mean ± SEM. *p < .05 vs. control.

Fig. 4.

Viability (A) and LDH leakage (B) following exposure to various rock dusts and silica in differentiated THP-1 cells. Values are expressed as mean ± SEM. *p < .05 vs. control.

Fig. 5.

Intracellular caspase-1 activity following 24 h exposure to various concentrations of rock dusts and crystalline silica in THP-1 cells. Values are expressed as mean ± SEM. *p < .05 vs. control.

Fig. 6.

Hierarchical cluster analysis of inflammatory cytokines (A), chemokines (B) and T_H2-cytokines (C) in THP-1 supernatants 24 h post-exposure to various rock dusts or crystalline silica.

Fig. 7.

LDH leakage in the supernatant (A) and intracellular caspase-1 activity (B) following 6 h of exposure to various rock dusts or silica in THP-1 cells pretreated with Bafilomycin A, Cytochalasin D or exposure medium with 0.1% DMSO. Values are expressed as mean ± SEM. *p < .05 vs. respective control.

Fig. 8.

The summary of the effects of Cytochalasin D and Bafilomycin A pretreatment on cytotoxicity (LDH leakage) and caspase-1 activation in PMA-differentiated THP-1 macrophages upon exposure to RD or Crystalline Silica.

Fig. 9.

The schematic illustrating the effect of Cytochalasin D and Bafilomycin A on caspase-1 activation in PMA-differentiated THP-1 macrophages upon exposure to treated marble (TM) OR crystalline silica.