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Pulmonary In Vitro Toxicity Assessment of Copper Carbonate Particles and Their Use as Outdoor Wood Preservatives
Citation:
Dreher, K., T. Luxton, M. Hartman, K. Belton, AND M. Kubo. Pulmonary In Vitro Toxicity Assessment of Copper Carbonate Particles and Their Use as Outdoor Wood Preservatives. North Carolina SOT, RTP, NC, October 15, 2018.
Impact/Purpose:
Research suggests that evaluation of the NP alone and not as a product may not be sufficient to determine their health risk. However, the results do demonstrate the ability of alternative assays to link physicochemical properties to potential adverse pulmonary health effects.
Description:
Nano\micro scale CuCO3·Cu(OH)2 (CuCO3) particles are being employed as a preservative for outdoor wood. This application raises the risk of dermal and/or inhalation exposures to nano/microparticles (NP) with unknown health effects. To address this uncertainty, research was conducted to assess the potential pulmonary toxicity of CuCO3 employed in outdoor wood preservation. CuCO3 underwent ball-milling to produce samples differing in particle size distribution which overlapped with commercial products containing nano and micro sized CuCO3, employed in outdoor wood preservation. Saw dust (SD) samples were obtained from untreated and commercially available CuCO3 treated outdoor wood. A thiobarbituric acid reactive substance (TBARS) assay was employed to assess the inherent oxidative reactivity of all samples examined in this research. TBARS analysis of milled CuCO3 samples demonstrated that samples with the smallest size distribution displayed the highest reactivity. CuCO3 NP treated outdoor wood SD displayed TBARS reactivity similar to CuCO3 ball-milled samples. SD from untreated outdoor wood showed no significant inherent oxidative reactivity. TBARS reactivity of treated SD samples supernatant was eliminated after filtration through a 3kDa filter but not a 0.2 µm filter. Human bronchial epithelial BEAS2B cells were exposed to milled and unmilled CuCO3 particles at a concentration range of 25 – 200 µg/ml as well as SD extracts at a concentration range of 2.75 – 88 mg/ml. Cytotoxicity was assessed using the WST1 assay at 22 hr post-exposure. Of the CuCO3 samples, the milled samples with the smallest size distribution were most cytotoxic to BEAS2B cells. The treated SD extracts were found to have the greatest cytotoxicity when compared to SD produced from untreated wood. qRT-PCR was performed to assess proinflammatory and oxidative gene expression at 3, 6, 12, 24, and 48 hr post-exposures to ball-milled CuCO3 NP, un-milled CuCO3 or SD extract samples. CuCO3 and SD extract exposure elicited proinflammatory gene expression of interleukin-6 (IL-6) and/or interleukin-8 (IL-8) and hemoxygenase-1 (HO-1), an indicator of oxidative stress. The magnitude of gene expression was generally higher in the milled CuCO3 versus the unmilled CuCO3. The time dependent gene expression profiles varied greatly between the CuCO3 samples and treated SD samples. These findings demonstrate nano\micro scale CuCO3 particles employed as wood preservatives have inherent reactivity which correlated with human airway epithelial cell cytotoxicity. The difference in cytotoxicity and gene expression profiles between the CuCO3 NP and SD samples suggest that evaluation of the NP alone and not as a product may not be sufficient to determine their health risk. However, the results do demonstrate the ability of alternative assays to link physicochemical properties to potential adverse pulmonary health effects.