CEMENT DUST INDUCED-IMMUNOTOXICOLOGICAL AND CELLULAR RESPONSES IN LUNG CELLS

Abstract

Recent epidemiological studies revealed that exposure to cement dust is associated with development of laryngeal cancer and other metal related diseases. The mechanism of injury to lung cells and disease development by this particle is still unclear, In this study, the effects of cement dust on some cellular functions in lung cells were investigated, Cement Dust samples from Nigeria (CDN) and United States of America (CDUSA) and Clinker from Nigeria (CCN) were used in the study. Alveolar type II epithelial cells (A549) and alveolar macrophages (NR8383) exposed to Cement Dust Extracts (C IL 2-20%) or cultured in Direct Contact (DC, 10.200g) with cement dust were compared with cells cultured in fresh culture medium (control) only. Total mercury, copper, chromium, cadmium, nickel, manganese, lead, iron levels in CCM CDN, and CDUSA were determined using graphite furnace atomic absorption spectrophotometry, while zinc and calcium were determined by flame atomic absorption spectrophotometry, and chromium VI (CR(VI)) by spectrocolorimetry. Endocytosis of particles was assessed using transmission electron microscope. Cell viability was determined using 3-(4. 5. dirficthyl1hiazol-2-yl)-2, 5-diphenylictrazoliurn bromide assay, while eytotoxicity was assessed through leakage Lactate Dehydrogenase (LDH) using ELISA. Apoptosis (annexin.V-PI), intracellular Reactive Oxygen Species (iROS) generation and reduced glutathione (GSH) were assessed using flow cytometry, while in situ DNA strand breaks was determined by terminal deoxynucleotidyl transferase-dUTP nick end labeling (TUNEL) assay by fluorescence microscopy. Tumour necrosis factor-a (TNF -a), interleukin-1β (IL-1β) and macrophate inflammatry protein-2 (MIP.2) secretion from NR8383 supernatants were evaluated by ELISA. Data were analysed using Student's t-test at p = 0.05. Copper, Nickel and Manganese were significantly higher in CDUSA relative to CDN. Both total Cr (CDUSA, 597.5± 64.9 vs CDN, 91.7 ± I9.9 or CCN, 110.3 ± 403 µg/g) and Cr (Vi) (CDUSA, 153.3 ± 8.3 vs CDN, 11.5 0.04 or CCN, 5.8± 0.01 µg/g) were also higher in CDUSA than in CDN. Cadmium (CDUSA, 0.1 ± 0.01 vs CD N, 0.6 1 0.04 or CCNl, 0.5 ± 0+03 µg/g) was higher in both CDN and CCN. Mercury (CDUSA, 151.5 ± 18.6 vs CDN, 297.0 ± 38.1 or CCN; 344.0 ± 8.6 µg/g) was more in both CDN and CCN, while lead (CDUSA; 3.0 ± 0.6 vs CDN, 3.9 ± 0.6 or CCM 4.4 ± 0.5 µg/g) was only significantly higher in CCN. Alveolar epithelial cells internalised clinker predominantly at the membrane bound vacuoles, Dose-dependent decrease in viable cells was observed with tested cement dusts or clinker. Values obtained in CDE assays were not consistent with those of DC. The CDUSA induced more apoptosis (NR8383 = 70.4 %), DNA strand breaks, intracellular ROS generation (22%) and reduced GSH compared with control, which may be related to the significant Cr (VI) level in CDUSA. Increase in IL.Iβ and TNF-a secretion were consistent in both CDE and DC, while MIP-2 was significantly increased in cell as exposed to CDE and Clinker. Endocrosis of cement dust particles, oxidative stress induced-apoptosis, DNA- strand breaks and induction or pro-inflammatory cytokines may be the key mechanisms of cement dust immunotoxicity in lung cells. Key' Words: Cement dust-induced toxicity, Clinker, Cytokines, Alveolar epithelial cells Word Counts: 498