From the Cover: Thirdhand Cigarette Smoke Causes Stress-Induced Mitochondrial Hyperfusion and Alters the Transcriptional Profile of Stem Cells.

Thirdhand cigarette smoke (THS) was recently recognized as an environmental health hazard; however, little is known about it effects on cells. Mitochondria are sensitive monitors of cell health and report on environmentally induced stress. We tested the effects of low levels of THS extracted from terry cloth on mitochondrial morphology and function using stem cells with well-defined mitochondria. Concentrations of THS that did not kill cells caused stress-induced mitochondrial hyperfusion (SIMH), which was characterized by changes in mitochondrial morphology indicative of fusion, increased mitochondrial membrane potential (MMP), increased ATP levels, increased superoxide production, and increased oxidation of mitochondrial proteins. SIMH was accompanied by a decrease in Fis1 expression, a gene responsible for mitochondrial fission, and a decrease in apoptosis-related genes, including Aifm2, Bbc3, and Bid There was also down regulation of Ucp2, Ucp4, and Ucp5, genes that decrease MMP thereby reducing oxidative phosphorylation, while promoting glycolysis. These effects, which collectively accompany SIMH, are a prosurvival mechanism to rescue damaged mitochondria and protect cells from apoptosis. Prolonged exposure to THS caused a reduction in MMP and decreased cell proliferation, which likely leads to apoptosis.

Cytotoxicity of Thirdhand Smoke and Identification of Acrolein as a Volatile Thirdhand Smoke Chemical That Inhibits Cell Proliferation.

Thirdhand smoke (THS) is a mixture of chemicals that remain on indoor surfaces after smoking has ceased. These chemicals can be inhaled, ingested, or absorbed dermally, and thus could impact human health. We evaluated the cytotoxicity and mode of action of fresh and aged THS, the toxicity of volatile organic chemicals (VOCs) in THS, and the molecular targets of acrolein, a VOC in THS. Experiments were done using mouse neural stem cells (mNSC), human pulmonary fibroblasts (hPF), and lung A549 epithelial cells. THS-exposed cotton cloth was extracted in Dulbecco's Eagle Medium and caused cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. THS extracts induced blebbing, immotility, vacuolization, cell fragmentation, severing of microfilaments and depolymerization of microtubules in mNSC. Cytotoxicity was inversely related to headspace volume in the extraction container and was lost upon aging, suggesting that VOCs in THS were cytotoxic. Phenol, 2',5'-dimethyl furan and acrolein were identified as the most cytotoxic VOCs in THS, and in combination, their cytotoxicity increased. Acrolein inhibited proliferation of mNSC and hPF and altered expression of cell cycle regulatory genes. Twenty-four hours of treatment with acrolein decreased expression of transcription factor Dp-1, a factor needed for the G1 to S transition in the cell cycle. At 48 h, WEE1 expression increased, while ANACP1 expression decreased consistent with blocking entry into and completion of the M phase of the cell cycle. This study identified acrolein as a highly cytotoxic VOC in THS which killed cells at high doses and inhibited cell proliferation at low doses.

Thirdhand smoke: Chemical dynamics, cytotoxicity, and genotoxicity in outdoor and indoor environments.

We tested the toxicity of thirdhand smoke (THS) using two controlled laboratory exposure scenarios and low levels of THS. One exposure modeled THS in a car parked outdoors, while the second modeled THS in a room without sunlight. The fabrics were exposed to cigarette smoke and then extracted in culture medium. Concentrations of nicotine, nicotine related alkaloids, and tobacco specific nitrosamines (TSNAs) were determined in fresh and aged extracts. The concentration of TSNAs increased with aging in the indoor experiment. THS extracts were used for cytotoxicity testing using mouse neural stem cells (mNSC), human dermal fibroblasts (hDF) and human palatal mesenchyme cells (hPM). Extracts from the car experiment inhibited mNSC proliferation in a live cell imaging assay and induced single strand DNA breaks in mNSC and hDF. In the indoor experiment, THS extracts made with medium containing serum proteins were significantly more toxic than extracts made with basal medium, and mNSC and hPM were more sensitive than hDF. These data indicate that: (1) aging of THS chemical differs on different fabrics and differs with and without sunlight; (2) very few cigarettes are sufficient to produce a toxic THS residue; and (3) protein enhances the efficiency of extraction of cytotoxic chemicals.

Thirdhand cigarette smoke: factors affecting exposure and remediation.

Thirdhand smoke (THS) refers to components of secondhand smoke that stick to indoor surfaces and persist in the environment. Little is known about exposure levels and possible remediation measures to reduce potential exposure in contaminated areas. This study deals with the effect of aging on THS components and evaluates possible exposure levels and remediation measures. We investigated the concentration of nicotine, five nicotine related alkaloids, and three tobacco specific nitrosamines (TSNAs) in smoke exposed fabrics. Two different extraction methods were used. Cotton terry cloth and polyester fleece were exposed to smoke in controlled laboratory conditions and aged before extraction. Liquid chromatography-tandem mass spectrometry was used for chemical analysis. Fabrics aged for 19 months after smoke exposure retained significant amounts of THS chemicals. During aqueous extraction, cotton cloth released about 41 times as much nicotine and about 78 times the amount of tobacco specific nitrosamines (TSNAs) as polyester after one hour of aqueous extraction. Concentrations of nicotine and TSNAs in extracts of terry cloth exposed to smoke were used to estimate infant/toddler oral exposure and adult dermal exposure to THS. Nicotine exposure from THS residue can be 6.8 times higher in toddlers and 24 times higher in adults and TSNA exposure can be 16 times higher in toddlers and 56 times higher in adults than what would be inhaled by a passive smoker. In addition to providing exposure estimates, our data could be useful in developing remediation strategies and in framing public health policies for indoor environments with THS.

Adaptation of stem cells to 96-well plate assays: use of human embryonic and mouse neural stem cells in the MTT assay.

Human embryonic stem cells (hESC) are difficult to adapt to 96-well plate assays, such as the MTT assay, because they survive best when plated as colonies, which are not easily counted and plated accurately. Two methods were developed to address this problem. In the first, ROCK inhibitor (ROCKi) was used, which allows accurate counting and plating of single hESC. In the second, small colonies were plated without ROCKi but with adaptations for accurate counting and plating. The MTT assay was also adapted for use with mouse neural stem cells. These methods allow the MTT assay to be conducted rapidly and accurately with high reproducibility between replicate experiments. When screening volatile chemicals in a 96-well plate, vapor effects may occur and dose ranges must be carefully defined. The methods were validated using the NIH assay guidance tool. These methodss could readily be translated to other 96-well plate assay.

Comparison of electronic cigarette refill fluid cytotoxicity using embryonic and adult models.

Electronic cigarettes (EC) and refill fluids are distributed with little information on their pre- and postnatal health effects. This study compares the cytotoxicity of EC refill fluids using embryonic and adult cells and examines the chemical characteristics of refill fluids using HPLC. Refill solutions were tested on human embryonic stem cells (hESC), mouse neural stem cells (mNSC), and human pulmonary fibroblasts (hPF) using the MTT assay, and NOAELs and IC(50)s were determined from dose-response curves. Spectral analysis was performed when products of the same flavor had different MTT outcomes. hESC and mNSC were generally more sensitive to refill solutions than hPF. All products from one company were cytotoxic to hESC and mNSC, but non-cytotoxic to hPF. Cytotoxicity was not due to nicotine, but was correlated with the number and concentration of chemicals used to flavor fluids. Additional studies are needed to fully assess the prenatal effect of refill fluids.