Effects of Cigarette Smoke Residues from Textiles on Fibroblasts, Neurocytes and Zebrafish Embryos and Nicotine Permeation Through Human Skin

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JOURNAL TITLE: International journal of hygiene and environmental health

USER JOURNAL TITLE:International journal of hygiene and environmental health.

ARTICLE TITLE: Effects of cigarette smoke residues from textiles on fibroblasts, neurocytes and zebrafish embryos and nicotine permeation through human skin

ARTICLE AUTHOR: Hammer

VOLUME: 214

ISSUE: 5

MONTH: September

YEAR: 2011

PAGES: 384-391

ISSN: 1438-4639

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This material may be protected by copyright law (Title 17 U.S. Code) International Journal of Hygiene and Environmental Health 214 (2011) 384-391

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Effects of cigarette smoke residues from textiles on fibroblasts, neurocytes and zebrafish embryos and nicotine permeation through human skin Timo R. Hammer*, Kirsten Fischer, Marina Mueller, Dirk Hoefer Hohenstein Institutes, Institute for Hygiene and Biotechnology, Schloss Hohenstein, 74357 Boennigheim, Germany

ARTICLE INFO ABSTRACT

Article history: Toxic substances from cigarette smoke can attach to carpets, curtains, clothes or other surfaces and Received 8 February 2011 thus may pose risks to affected persons. The phenomenon itself and the potential hazards are discussed Received in revised form 5 April 2011 controversially, but scientific data are rare. The objective of this study was to examine the potential of Accepted 29 April 2011 textile-bound nicotine for permeation through human skin and to assess the effects of cigarette smoke extracts from clothes on fibroblasts, neurocytes and zebrafish embryos. Tritiated nicotine from contami- Keywords: nated cotton textiles penetrated through adult human full-thickness skin as well as through a 3D in vitro Cigarette skin model in diffusion chambers. We also observed a significant concentration-dependent cytotoxic- ETS Tobacco ity of textile smoke extracts on fibroblast viability and structure as well as on neurocytes. Early larval Cancer tests with zebrafish embryos were used as a valid assay for testing acute vertebrate toxicity. Zebrafish Risk assessment development was delayed and most of the embryos died when exposed to smoke extracts from textiles. Third-hand smoke Our data show that textiles contaminated with cigarette smoke represent a potential source of nicotine uptake and can provoke adverse health effects. © 2011 Elsevier GmbH. All rights reserved.

Introduction ing and other surfaces in the environment of the smoker. Also the smoker's breath is a source of tobacco-related volatile organic Many studies show the negative health effects of tobacco smoke compounds (Buszewski et al., 2009). Sleiman et al. (2010) and on the smokers themselves and their environment with tobacco Petrick et al. (2011) showed the formation of toxicants by surface- smoke containing more than 4000 chemicals including numerous mediated reactions of nicotine with other substances and thus the known and suspected carcinogens (Rodgman and Perfetti, 2008). potential hazard of residual tobacco smoke contamination, which While the smokers inhale mainstream smoke, people around them could especially be dangerous for infants and children (Matt et al., are exposed to second-hand or sidestream smoke, which con- 1999, 2004; Winickoff et al., 2010). While particle size and amount tains even higher concentrations of many tobacco components (Becquemin et al., 2010) as well as volatile organic compounds than mainstream smoke (Wroth, 1989; Wong et al., 2004). Envi- (Ueta et al., 2010) of third-hand smoke were determined, to our ronmental Tobacco Smoke (ETS) is a combination of both which knowledge, a direct effect of smoke residues from textiles on liv- becomes rapidly diluted in ambient air. The effects of passive smoking tissues or cells has not been proven so far. These data could ing on human health were studied extensively (Argacha et al., 2008; be very important, however, as sidestream smoke was shown to Bakirtas, 2009; Flouris, 2009; Gill et al., 2010; Kraemer et al., 2004; have effects on structure and function of fibroblasts with impacts Lefcoe, 1986; Magnussen et al., 1993; Otsuka et al., 2001; Raupach on wound healing and tissue repair (Wong et al., 2004). Further- et al., 2006; Simoni et al., 2007; Wiedemann et al., 1986; Yarlioglues more, the enzyme activity of human lung fibroblasts is affected by et al., 2010) and a recent global study found that 600,000 people mainstream cigarette smoke extract (Ning et al., 2007). die because of passive smoking each year (Oberg et al., 2010). The nicotine levels of tobacco products vary (Gritz et al., 1981; During the last few years, another part of cigarette smoke - Hecht et al., 1975; Squier, 1986). For instance, the smoke of one third -hand smoke (THS) - has moved to the focus of attention and is cigarette usually contains between 0.4 and 3.0 mg nicotine. Since discussed controversially (Dreyfuss, 2010; Tuma, 2010; Winickoff 2004 the maximum nicotine content per cigarette in the Euro- et al., 2009). The term describes the residual part of the smoke pean Union is restricted to 1.0 mg. Various acute effects of nicotine which is not inhaled but adheres to curtains, carpets, hair or cloth- via nicotinic acetylcholine receptors could be shown for example on skin (Grando, 2001), oral epithelial cells or keratinocytes (Arredondo et al., 2001, 2005, 2006, 2007, 2008; Lee et al., 2005) * Corresponding author. Tel.: +49 7143 271 410; fax: +49 7143 271 94433. as well as direct effects on dermal fibroblasts (Choi et al., 2010) E-mail address: [email protected] (T.R. Hammer). and epithelial cells (Conklin et al., 2002). Other studies indicate

1438-4639/$ - see front matter © 2011 Elsevier GmbH. All rights reserved. doi:10.1016/j.ijheh.2011.04.007 T.R. Hammer et al. / International Journal of Hygiene and Environmental Health 214 (2011) 384-391 385 direct impacts of nicotine on nervous system development by the any residual air underneath the skin. A circular swatch of cot- affection of axonal outgrowth of spinal motoneurons in zebrafish ton textile (diameter: 1 cm) endowed with 3H-labelled nicotine (Svoboda et al., 2002; Welsh et al., 2009), cholinoceptive neurons (85 Ci/mmol, 25 pmol/textile patch, dried) was placed in the mid- in developing chick tectum (Torrao et al., 2003) or hippocampal dle of each skin sample. 50 pA of PBS (pH 7.4) or artificial sweat H19-7 cells (Shin-young et al., 2007). (pH 5.5) were added to the textile swatches to dissolve the dried Although numerous adverse effects of tobacco smoke on the skin nicotine. The Franz-type diffusion cell was closed with parafilm to are known (Krug et al., 2004), the effects of pure nicotine on skin and prevent drying-out of the system. After incubation of the whole wound healing are even more controversial (Forrest et al., 1987; diffusion cell for 24 h at 37 °C, the textile swatches were removed Misery, 2004). For example, the alkaloid was shown to promote and the residual textile-bound nicotine was eluted for 30 min at wound healing at low concentrations (Jacobi et al., 2002; Morimoto 37 °C with gentle agitation in 6.5 ml of PBS. This solution, as well et al., 2008), to induce angiogenesis (Heeschen et al., 2001; Jacobi as the PBS from the lower chamber of the Franz-type diffusion cell, et al., 2002) or to increase cutaneous blood flow (Sorensen et al., were transferred directly to liquid scintillation counting (LSC) to 2009; Usuki et al., 1998) when applied in its pure form, not as a determine the percentage of residual textile-bound nicotine and tobacco ingredient. On the other hand, Inaloz et al. (2000) found of the nicotine which completely permeated through the skin. The considerable teratogenic effects of nicotine on new born rat skin. skin sample was homogenized, diluted in PBS and also transferred The ability of nicotine to penetrate skin (Farahmand and Maibach, to LSC. In an additional assay, 1 ml of TSE-10 was added onto the 2009) and mucosa (Squier, 1986) is used for smoking replacement skin sample instead of the textile swatch and the PBS in the lower therapies with nicotine-containing patches or chewing gums. chamber was replaced by 12 ml of DMEM cell culture medium. After In our study we intended to examine the effects of cigarette incubation for 24 h at 37 °C, the medium from the lower chamber smoke residues from textiles on skin and living cells. As the adhe- was used for the fibroblast cytotoxicity assay. To repeat the exper- sion and dehesion of smoke components to and from textiles iments with a more susceptible skin equivalent, a 3D in vitro skin strongly depend on the material the fabric is made of (Ueta et al., model was produced as described elsewhere (Mertsching et al., 2010), we decided to use standardized cotton cloth since cotton is 2008) and skin penetration assays were performed as described the predominantly used cloth worldwide. Artificial human sweat, a above. common solvent for testing textile-associated toxicity, was used for extracting cigarette smoke from the treated cotton. Radioactively labelled nicotine was used for tracer experiments to evaluate the Liquid scintillation counting (LSC) risk potential of nicotine transfer and penetration capacity from contaminated textile patches to adult human full-thickness skin Samples (eluted residual textile-bound nicotine, PBS from lower and to 3D in vitro skin model. Subsequently, the effects of textile chamber in Franz-type diffusion cell and homogenized skin in PBS) sweat extracts (TSE) on fibroblasts and neurocytes were docu- were diluted with deionized water to a final volume of 6 ml in a mented. To evaluate vertebrate toxicity, experiments on the impact 20 ml plastic scintillation vial. 8 ml of scintillation cocktail (Aqua- of TSE on zebrafish development were conducted. Light, Hidex) were added to each sample and the mixture was gently inverted to obtain a homogenous suspension. Subsequently, the samples were placed in a liquid scintillation counter (Hidex 300 Materials and methods SL) and measured with 120 s detection time for 3 H -detect io n. The obtained data were processed using MikroWin software (Hidex). Preparation of textile sweat extracts (TSE)

Swatches of standard cotton cloth (log) were fixed inside a Fibroblast cytotoxicity assay 51 glass flask and slightly moistened with deionized water. The flask was arranged upside-down on 1 cm thick plastic strips to Murine fibroblasts (ATCC#CCL1 clone L929) were grown in cell obtain a window for inserting the cigarettes. 1, 5 or 10 commer- culture flasks with DMEM culture medium at standard culture cially available cigarettes (smoke of each containing 10 mg tar, conditions (37 °C with 5% CO2 in a 'Cytoperm 2' CO2-incubator). 0.8 mg nicotine and 10 mg CO) were lit up and put immediately The cytotoxicity assay was performed according to DIN EN ISO under the glass flask. As the whole experimental set-up was placed 10993-5. In brief, confluent cells were detached from the cell cul- in a room free of air turbulences, the smoke remained within ture flask, adjusted with fresh DMEM to a final concentration of the glass bell. After 60 min, the contaminated textiles were taken 1 x 105 cells/ml and transferred to a 96-well culture plate with out, dried and subsequently conducted to extraction with artifi- 50µl per well. After 30 min of incubation at 37 °C and 5% CO2, cial human sweat (0.5 g/11-histidine, 5 g/INaCI, 2.2 g/1NaH2PO4, pH 100 pA of the sample solutions (TSE-1, TSE-5, TSE-10, 12 mM nico- 5.5; 10 ml/g textile) for 24 h at 37 °C with gentle agitation. Artificial tine in artificial sweat, medium from skin penetration assay with sweat is the standard solvent used in textile toxicology testing pro- TSE-10) were added to each well. Sample concentration was var- cedures. Finally, the textile sweat extracts from textiles exposed to ied to obtain final sample concentrations of 33.3%, 22.2%, 14.8% and 1 cigarette (TSE-1), 5 cigarettes (TSE-5) and 10 cigarettes (TSE-10) 9.9%. 5% DMSO was used as a negative control. Each sample con- were obtained, adjusted to pH 7.4 and frozen at -26 °C until use. In centration was tested at least in 3 independent experiments. After addition, a non-treated textile was extracted likewise with artificial addition of the sample solutions, the cells were incubated for 72 h sweat as a negative control for subsequent experiments (TSE-0). as described above. To measure cell viability, a BCA-assay was conducted. The medium was removed from the cells and the cells were Full-thickness skin penetration assay washed 3 times with PBS (pH 7.4). Subsequently, the cells were frozen at -28 °C for 30 min and thawed at 37 °C with gentle agita- Fresh adult full-thickness donor skin removed from the breast tion. The freezing/thawing procedure was repeated twice. BCA-A of a female patient during plastic surgery was kept in PBS at 4 °C and BCA-B reagent were mixed 1:50 and 200 pA of the mixture until use. For the penetration assays, circular pieces of the skin were added to each well. The cells were mechanically detached (diameter: 3 cm) were fixed in Franz-type diffusion cells with the and mixed with the reagent using a pipette tip. After a 30 min epidermis upwards. The lower chamber was completely filled with incubation step at 60 °C and subsequent chilling at room tempera- 12 ml of phosphate buffered saline (PBS, pH 7.4) without leaving ture, absorption at 540 nm was determined with a multiwell plate 386 T.R. Hammer et al. /International Journal of Hygiene and Envonmental Health 214 (2011) 384-391 reader. Cell growth inhibition was defined by the absorption ratio of (sample - blank)/(solvent control - blank). 100 90 Microscopy 80 Prior to seeding the cells, a sterile glass coverslip was placed on 70 the bottom of each well in a 6-well culture plate. Primary human 60 fibroblasts (9 x 104 cells/ml) were seeded into the wells and fed iic 50 with DMEM culture medium for 24 hat standard culture conditions. E 40 After 24 h the medium was replaced by fresh DMEM supplemented with TSE-0, 1.5 mM nicotine in artificial sweat or TSE-10, respec- cn 30 tively to a final concentration of 33.3%. After 6 h of incubation 20 at standard culture conditions, the coverslips were fixed in 4% 10 14.8% buffered paraformaldehyde and incubated at 4 °C for at least 24 h. 22.2% itt^, For labelling the cells, coverslips were washed three times with TSE-1TSE-5 33.3% 6, TSE-10 0 PBS, incubated for 5 min in PBS/0.5% Triton X-100 and covered with Nic phalloidin-rhodamine (1:200 in PBS) for 30 min in the dark. The Med coverslips were washed three times with 10 p.M 4',6-diamidino-2- sample FDC phenylindole (DAPI) in PBS and washed twice in PBS. Finally, the Fig. 1. Cell viability of L929 fibroblasts exposed to different concentrations of TSE- samples were fixed in mounting medium on glass slides for subse- 1, TSE-5, TSE-10, nicotine (Nic) and to culture medium from Franz-type diffusion quent fluorescence microscopy on an Olympus AX-70 fluorescence cell (Med FDC). All samples affected cell viability negatively in a dose-dependent microscope. manner. The threshold for cytotoxicity is defined as cell survival of 70% according to DIN EN ISO 10993-5. Thus, TSE-5 and TSE-10 were cytotoxic in all concentrations tested whereas nicotine solution was only cytotoxic in the 33.3% concentration. Neurite growth assay

To determine the influence of nicotine and TSE-10 on neurite nicotine content in the textile, in the skin sample as well as in the outgrowth, the tecta of 6 day old sacrificed chick embryos were medium underneath the skin was determined using liquid scintil- isolated, fragmented mechanically and trypsinized. The trypsinized lation counting (LSC). The results are shown in Table 1. neurocytes were washed and diluted in DMEM culture medium Permeation of nicotine through the skin could be shown con- to a final concentration of 1 x 105 cells/ml. 400 FA of the cell sus- sistently in all samples, penetration rate, however, was strongly pension were added into each well of collagen-coated 24-well dependent on the solvent used. With PBS, more nicotine was cell culture plates and incubated until the neurocytes adhered to detached from the textile and penetrated the skin. About 50% of the bottom. Sample solutions of nicotine in artificial sweat and total nicotine was found in the lower diffusion chamber underneath TSE-10 were added in different concentrations and the cells were the skin. More nicotine (54%) remained on the textile if artificial incubated at standard culture conditions for 72 h. After incuba- sweat was used as a solvent and only a small amount (5.7%) perme- tion, the cells were washed three times with PBS and fixed with ated through the skin into the medium. The variance of the obtained 4% buffered paraformaldehyde at 4 °C. Neurites were stained with data was significantly higher with PBS than with artificial sweat. May-Gruenwald staining solution and photographed on an Olym- Using a similar 3D in vitro skin model, we found that most of the pus IX-70 inverted microscope. The pictures were used for cell nicotine (90-99%) from contaminated textile swatches permeated counting and neurite length measurements with Image.] software through the artificial skin into the lower diffusion chamber under- and the obtained data were evaluated statistically. neath the skin, whereas only little nicotine (1-10%) remained in the textile (data not shown). Zebrafish early larval tests Fibroblast viability Female and male zebrafish (Danio rerio, strain 'WIK') were kept together in a standard aquarium (1121) at 28 °C and 14 h light/day Textile sweat extracts (TSE) from cotton textiles exposed to with internal filtering. To harvest fertilized zebrafish eggs, a cus- smoke from 1, 5 or 10 cigarettes in a practical approach were used tomized trap was placed into the aquarium 14 h prior to the for BCA cytotoxicity assays with L929 fibroblasts. In addition, we experiments. Fertilized eggs were collected from the trap 1 h after also tested the medium which was underneath the TSE-treated laying and washed three times with E3 zebrafish embryo medium skin model in a Franz-type diffusion cell for cytotoxicity accord- (Brand et al., 2002). 2 eggs were placed together with 1 ml of E3 ing to DIN EN ISO 10993-5. A significant dose-dependent cytotoxic medium or E3 medium supplemented with 10% of TSE-0, 12 mM effect on L929 fibroblasts could be observed. The results are sum- nicotine in AS, TSE-5 or TSE-10 in each well of a 24-well culture marized in Fig. 1. According to the mentioned standard, an extract plate and incubated at 28 °C. Sample medium was replaced every is cytotoxic if cell survival is below 70% at the 33.3% sweat extract day. Photographs were taken after certain periods of time on an concentration. Thus, all textile sweat extracts as well as the medium inverted microscope. from underneath the treated skin in the Franz-type diffusion cell were cytotoxic in our experiments. The effect was dose-dependent Results with an increasing toxicity from TSE-1 (cell survival 68% ± 3.61) to TSE-5 (cell survival 13% ± 0) and TSE-10 (cell survival 4.3% ± 1.51). Permeation of nicotine through the skin TSE-5 and TSE-10 were toxic to the lowest concentration tested (14.8%). The toxic effect of nicotine alone in a high concentration To evaluate the penetration capacity of nicotine transfer from (12 mM in artificial sweat) was assessed for comparison and was clothes into and its permeation through the skin, cotton swatches also significant (cell survival 29% ± 9.99 with the 33.3% concentra- treated with 3H-labelled nicotine (dissolved in PBS or artificial tion). The medium collected from underneath the skin samples in sweat) were incubated on human adult full-thickness skin in a the nicotine permeation assays showed a cytotoxic effect similar to Franz-type diffusion cell. After 24 h of incubation the remaining TSE-1. T.R. Hammer et al. / International Journal of Hygiene and Environmental Health 214 (2011) 384-391 387

Table 1 Mean permeation of 3H- nicotine (37 Bq/pmol) from textile swatches through adult human donor skin.

Artificial sweat PBS

3 H- signal [13q] Nicotine [pmol] % of total nicotine 3H-signal [13q] Nicotine [pmol] % of total nicotine

Textile 504± 124.3 13.6± 3.4 54.3± 13.4 195.7± 47.1 5.3± 1.3 21± 5.2 Skin 372± 84.5 10.0± 2.3 40.0± 9.5 271.7± 178.2 7.3± 4.8 29.3± 19.3 Diffusion chamber 54± 44.1 1.5± 1.2 5.7± 4.5 461.7± 207.0 12.5± 5.6 49.7± 22.3

Fibroblast structure following 20 h with the heart beat of the treated fish being significantly lower than in the control group. In the late phase, prior to TSE-10 (33.3%) was applied on human fibroblasts for 6 h to hatching, most of the smoke extract- and nicotine-treated embryos assess putative effects on cell morphology and structure prior to died off. Throughout the assays, TSE-1 had no apparent effect on cell death. Representative pictures are shown in Fig. 2. Fibrob- zebrafish development. Fig. 4 gives an overview of the zebrafish lasts exposed to TSE-10 were significantly smaller in size and more embryo experiments. separated from each other compared to the control with 33.3% of uncontaminated artificial sweat (TSE-0). In addition, fibroblasts Discussion in the TSE-10-assays were less elongated than the control cells. In about 1% of the TSE-10-exposed human fibroblasts we found Several studies show the toxicity of cigarette smoke inhaled a nicotine-driven formation of vacuoles. All fibroblasts grown in directly by the smoker or passively. The phenomenon and the medium with TSE-10 showed an accumulation of black particles hazards of smoke attached to surfaces like curtains, carpets or inside the cells, which were likely to be particulate matter from the clothes are discussed controversially and scientific data are rare. cigarette smoke. As cell shape was clearly affected by TSE-10, we Here we show for the first time the toxic effects of cigarette smoke looked for changes in the actin-cytoskeleton in detail. No appar- residues dissolved from cotton textiles on fibroblasts, neurocytes ent alterations in the stress fibre pattern could be observed in the and zebrafish embryos and the permeation of textile-bound nico- TSE-treated cells compared to the control. tine through human skin. Our results imply that about half of the nicotine bound to cloth- Neurotoxicity ing and dissolved by skin sweat penetrates into and permeates through the adult skin. In this respect, our data are consistent A neurite growth assay was run with avian tectum neurons with the nicotine permeation values of the full-thickness skin exposed to TSE-0 as a control, nicotine in artificial sweat and model published by Ackermann et al. (2010) who found perme- TSE-10. Cells died off completely when exposed to 33.3% and ation of 46.3% ± 6.52 of total nicotine dissolved in PBS after 6 h and 22.2% artificial sweat/nicotine or 33.3%, 22.2% and 14.8% TSE-10, 70.4% ± 4.3 after 26 h in a Franz-type diffusion cell. It is known that respectively. Applying lower concentrations, neurite length could penetration rates of substances depend on the solvent used which is be measured in 100 individual cells per sample. Medium neurite also true for our data. Although it is known that most in vitro-based length and statistical length distribution were determined. The permeability models underestimate the in vivo data up to 10,000 - results are shown in Fig. 3. Our data show that neurocytes are sig- fold, especially for nicotine (Farahmand and Maibach, 2009), we nificantly affected by nicotine and especially by TSE-10. Medium used a low nicotine concentration (about 32 pmol nicotine/cm2 neurite length of the AS-nicotine-exposed cells was reduced to cotton textile) for the permeation experiments. In comparison, 62.7% (with 14.8% of nicotine-solution) and 36.9% (with 9.9% of common nicotine swatches in smoking replacement therapies con- nicotine solution) compared to the control (AS without nicotine). tain up to 25 mg (-154 nmol) nicotine. In our study, the most The effect of TSE-10 was stronger with a neurite length reduction realistic scenario for cigarette smoke residuals exposure to skin of 23.0% in the lowest concentration tested (9.9% TSE-10) and a from clothes is with human donor skin and artificial sweat as a complete cell death using higher TSE-10 concentrations. nicotine solvent. As about 50% of the nicotine entered or permeated the skin, we assume that tobacco smoke residue on clothes Effects on zebrafish development is a potential source of nicotine uptake. We obtained significantly higher permeation rates with a 3D in vitro skin model (data not Zebrafish early larval tests were performed. Fertilized zebrafish shown) which may be explained by differences in structure of the eggs were exposed to artificial sweat with nicotine as well as to stratum corneum, which is known to form a rate-controlling barrier 10% TSE-0, TSE-1, TSE-5 or TSE-10 and compared to a control in for diffusion of most compounds. normal embryo growth medium. The developmental stages were Forthetoxicityexperiments,wegeneratedsmoke- determined according to Kimmel et al. (1995). During the first 20 h contaminated cotton textiles in a realistic practical approach after fertilization there were no apparent differences between TSE- rather than producing defined third-hand smoke with a constant exposed and control embryos, but the number of coagulated eggs source of oxygen. The extracted smoke residues from the prepared significantly increased in the presence of TSE-5 and TSE-10 dur- textiles were toxic to fibroblasts and neurocytes. Fibroblast via- ing the following 24 h. Development of the surviving embryos was bility and structure was affected significantly, a finding which is delayed with TSE-5 and TSE-10 compared to the control with TSE-0. consistent with Wong et al. (2004). On the other hand, the fibrob- The delay in development was clearly visible during the pharyngula lasts in our TSE-10-assays were less elongated than the control period, defined by Kimmel et al. (1995), as the onset of melanogen- cells, which is contradictory to the above mentioned data from esis and pigmentation was later in the treated embryos and during 2004. Vacuolization was already shown in human keratinocytes the hatching period with the control embryos hatching earlier than exposed to nicotine by Theilig et al. (1994) and one decade later, the embryos treated with smoke extracts. In addition to the devel- the same effect was revealed in chicken primary fibroblasts with opmental delay, metabolic effects of smoke extract exposure could second-hand smoke as a toxicant (Wong et al., 2004). We were able be detected. After 30-35 h, a regular heart beat could be detected in to confirm the nicotine-driven formation of vacuoles in human all fish with the heart rate being increased in the smoke extract- and fibroblasts and found the same effect stimulated by TSE-10 in nicotine-treated group. Interestingly, this effect inverted during the about 1% of the exposed fibroblasts. We did not observe alterations 388 T.R. Hammer et al. /International Journal of Hygiene and Environmental Health 214 (2011) 384 -391

Fig. 2. Cell morphology of human fibroblasts exposed to 33.3% TSE-0, nicotine or TSE-10 for 6 h. (A-C) Cells exposed to TSE-0 (A), 1.5 mMnicotine in artificial sweat (B) and TSE-10 (C). F-actin labelled with phalloidin-rhodamine, nuclei labelled with DAPI. Scale bars (A-C) = 250 p.m. Vacuolization is apparent in nicotine-exposed cells. TSE-10 treated cells are clearly affected in size and cell density. (D + E) Detailed representative pictures of cells exposed to 33.3% TSE-10. Accumulation of blackparticulate matter (D, arrow heads) in all cells and vacuolization (E, arrows) in about 1% of the cells were typical effects of TSE-10-treatment. Scale bars (D; E)= 25 p.m. in the stress fibre pattern and the migration potential of the cells with implications for alterations in the nervous system (Berger exposed to the textile smoke extracts, although second-hand et al., 1998; Roy et al., 2002; Slotkin, 1998; Tomassini et al., 2007; smoke has been shown to increase stress-fibre formation in fibrob- Touiki et al., 2007). With our findings we add the aspect of cigarette lasts (Wong et al., 2004). Fibroblast propagation and migration are smoke residue from textiles as a potential neurotoxic agent, which crucial for wound healing. Several studies on the effect of active might be critical for example for children sucking on clothes or and passive smoking on wound healing have been published so other textiles which were previously exposed to cigarette smoke. far, displaying contradictory results. In cutaneous injuries of rats Therefore, potential effects of such extracts on zebrafish embryo less fibroblasts were found when the animals were exposed to development were used for testing acute vertebrate toxicity. We nicotine (Biondo-Simoes et al., 2009) and in another study wound were able to show the acute toxicity of extracted cigarette smoke closure in mice exposed to sidestream smoke was significantly residues from textiles on zebrafish development. TSE- as well delayed compared to the control group (Wong et al., 2004). Jacobi as nicotine exposure resulted in an increased heart beat during et al. (2002) however, report the nicotine-driven acceleration of the first 35 h of incubation, followed by a decrease in heart beat angiogenesis and wound healing in diabetic mice. In our study, during the following 20 h and a high embryo mortality prior to the extracted smoke residuals had significant negative effects on hatching. This phenomenon might be explained by the initial the viability and cell structure of fibroblasts in a dose-dependent nicotine-driven stimulation of heart rate and the associated higher manner. Although we concentrated on nicotine in high doses as energy consumption and a subsequent lack of energy resulting in a positive control, one should be aware that nicotine is not the embryo mortality. only component of cigarette smoke which could explain cyto- and To conclude, people should be aware of the negative effects of neurotoxicity. Substantial affection of neural cells by nicotine alone cigarette smoke residues on various cell types, for example when as well as by tobacco smoke could be shown in various studies infants get into contact with contaminated textiles as a source of T.R. Hammer et al. / International Journal of Hygiene and Environmental Health 214 (2011) 384-391 389

A Neurite length -nicotine B Neurite length -T5E-10 GOO 700

600 500 a_ 733 500 15, 400 -= 400 300 1 En y,300

200 200

E 100 100

0 =f= Control AS Nic 14.8% Nit 9.9% Control TSE-0 TSE-109,9%

Sample Sample

Neurite length distribution -nicotine Neurite length distribution - TSE-10

100 100 90 80 80 --o- Control 70 60 -o-AS 60 -o- Control

40 -A- NIc 14.8% 50 -0- 1SE-0 40 -A- TSE-1 [I 9.9% -a- Nic 9.9% o_ 20 30 20 0 10 0 500 1.00D 1.500 0 Neurite length [pixels] 500 1000 1500 Length [pixels]

Fig. 3.Neurite growth assay with isolated chicken tectum neurons. (A) Effect of artificial sweat (AS) and 12 mM nicotine in AS (Nic) in two different concentrations(9.9% and 14.8%) on medium neurite length and on statistical neurite length distribution. (B) Effect of TSE-0 and 9.9% TSE-10 on medium neurite length and on statisticalneurite length distribution. Neurite length was measured in pixels on cell microscopy photographs of 100 neurons per sample. Nicotine and TSE-10 clearly inhibited neurite outgrowth in avian tectum neurocytes.

Co TSE-O Nic TSE-5 TSE-10

28 h

45 h

53 h

Fig. 4.Effects of TSE-0, 12 mM nicotine in AS (Nic , TSE-5 and TSE-10 on zebrafish embryo development compared to a negative control (Co). Nicotine, TSE-5and TSE-10 significantly delayed embryo development in the period from 25 to 53 h. Scale bar = 300 p.m. 390 T.R. Hammer et al. /International Journal of Hygiene and Environmental Health 214 (2011) 384 -391 smoke residues. While our study focuses on the acute toxicity, Gill, I., Karapinar, H., Yarlioglues, M., Ozdogru, I., Kaya, M.G., Yilmaz, A., Turgut, 0.0., chronic effects of smoke residue exposure still have to be studied. In Tandogan, I., Eryol, N.K., 2010. Acute effects of passive smoking on endothelial functions. Angiology. addition, the putative interactions of the tobacco components with Hecht, S.S., Ornaf, R.M., Hoffmann, D., 1975. Chemical studies on tobacco smoke. textile finishes or the fibre surfaces should also be considered. In XXXIII. N'-nitrosonornicotine in tobacco: analysis of possible contributing fac- the future, further research has to be done to clarify open issues and tors and biologic implications. Journal of the National Cancer Institute 54 (5), 1237-1244. to support the controversial discussion on the risks of third-hand Heeschen, C., Jang, J.J., Weis, M., Pathak, A., Kaji, S., Hu, R.S., Tsao, P.S., Johnson, F.L, smoke scientifically. Cooke, J.P., 2001. Nicotine stimulates angiogenesis and promotes tumor growth and atherosclerosis. Nature Medicine 7 (7), 833-839. Inaloz, H.S., Inaloz, S.S., Deveci, E., Eralp, A., 2000. Teratogenic effects of nicotine on Acknowledgments rat skin. Clinical and Experimental Obstetrics and Gynecology 27 (3-4), 241- 243. Jacobi, J., Jang, J.J., Sundram, U., Dayoub, H., Fajardo, L.F., Cooke, J.P., 2002. Nico- The authors thank Nadja Berner-Dannenmann and Justine tine accelerates angiogenesis and wound healing in genetically diabetic mice. 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