Thirdhand tobacco smoke: procedures to evaluate cytotoxicity in cell cultures

The risks associated to tobacco smoking are not ceased with smoke extinction. Many toxic compounds remain in the environment after the cigarette is extinguished and accumulated in the air or on surfaces. However, little is known about the risks of this exposure. The aim of this study was to evaluate procedures to collect thirdhand smoke (THS) and prepare the samples to perform three in vitro toxicity tests. Cellulose papers and cotton wipes were used to impregnate with nicotine solution and smoke cigarette in a chamber or in smoker's home. Samples were immersed in methanol or Dulbecco's modified Eagle's medium (DMEM) to expose Hep-2 cells. MTT, neutral red uptake (NRU) and trypan blue assays were performed. The concentration of nicotine in DMEM extract of THS in paper and cotton was similar to those in methanol extract (p>0.05). Alterations in the mitochondrial and lysosomal functions were found in both paper and cotton samples; however, the cytotoxic effect was not always observed. There was a decrease of 21-31% in MTT assay and 38-56% in NRU assay (p<0.003). There was a dose-response relationship between the amount of cigarettes and lysosomal viability; the correlation was higher for cotton samples (r=-0.843, p<0.001). As a dose-response relationship was found only in NRU assay, this test may be a more suitable choice rather than the MTT assay. Paper and wipe sampling can be reliable markers of tobacco smoke contamination. Moreover, these materials, if properly prepared, can be used as substrate providers to perform cellular assays.