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Assessing Toxicity and in Vitro Bioactivity of Smoked Cigarette Leachate Using Cell-Based Assays and Chemical Analysis

  • Elvis Genbo Xu
    Elvis Genbo Xu
    Department of Environmental Sciences, University of California, Riverside, Riverside, California 92521, United States
  • William H. Richardot
    William H. Richardot
    School of Public Health, San Diego State University, San Diego, California 92182, United States
    San Diego State University Research Foundation, San Diego, California 92182, United States
  • Shuying Li
    Shuying Li
    Department of Environmental Sciences, University of California, Riverside, Riverside, California 92521, United States
    More by Shuying Li
  • Lucas Buruaem
    Lucas Buruaem
    Department of Environmental Sciences, University of California, Riverside, Riverside, California 92521, United States
  • Hung-Hsu Wei
    Hung-Hsu Wei
    School of Public Health, San Diego State University, San Diego, California 92182, United States
    More by Hung-Hsu Wei
  • Nathan G. Dodder
    Nathan G. Dodder
    School of Public Health, San Diego State University, San Diego, California 92182, United States
    San Diego State University Research Foundation, San Diego, California 92182, United States
  • Suzaynn F. Schick
    Suzaynn F. Schick
    Department of Medicine, Division of Occupational and Environmental Health, University of California, San Francisco San Francisco, California 94143, United States
  • Thomas Novotny
    Thomas Novotny
    School of Public Health, San Diego State University, San Diego, California 92182, United States
    San Diego State University Research Foundation, San Diego, California 92182, United States
  • Daniel Schlenk
    Daniel Schlenk
    Department of Environmental Sciences, University of California, Riverside, Riverside, California 92521, United States
  • Richard M. Gersberg
    Richard M. Gersberg
    School of Public Health, San Diego State University, San Diego, California 92182, United States
  • Eunha Hoh*
    Eunha Hoh
    School of Public Health, San Diego State University, San Diego, California 92182, United States
    *E-mail: [email protected]. Phone: (619) 594-4671.
    More by Eunha Hoh
Cite this: Chem. Res. Toxicol. 2019, 32, 8, 1670-1679
Publication Date (Web):July 9, 2019
https://doi.org/10.1021/acs.chemrestox.9b00201
Copyright © 2019 American Chemical Society
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Abstract

Smoked cigarettes are the most prevalent form of litter worldwide, often finding their way into oceans and inland waterways. Cigarette smoke contains more than 4000 individual chemicals, some of them carcinogenic or otherwise toxic. We examined the cytotoxicity, genotoxicity, aryl hydrocarbon receptor (AhR), estrogen receptor (ER), and p53 response pathways of smoked cigarette leachate in vitro. Both seawater and freshwater leachates of smoked cigarettes were tested. Cytotoxicity and genotoxicity were negligible at 100 smoked cigarettes/L, while statistically significant AhR, ER, and p53 responses were observed in the extracts of both leachates, suggesting a potential risk to human health through exposure to cigarette litter in the environment. To identify responsible chemicals for the AhR response, an effect directed analysis approach was coupled with nontargeted chemical analysis based on comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC/TOF-MS). Eleven compounds potentially responsible for the AhR response were identified. Among them, 2-methylindole was partially responsible for the AhR response.

Introduction

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Smoked cigarettes (called “cigarette butts”) are one of the most prevalent forms of litter worldwide, often finding their way into our oceans and inland waterways. An estimated 5.7 trillion cigarettes are produced every year,(1) and between one-third and two-thirds of these are deposited each year into the environment.(2) Analysis of marine litter in the Mediterranean Sea found that cigarette butts accounted for 22.9% of all litter collected on the beach, the single most common form of litter categorized in the study.(3) Additionally, the Ocean Conservancy reported 1,863,838 cigarette butts were collected during their 2016 annual International Coastal Cleanup event, the single most collected item of the event.(4)
Cigarette filters are composed of cellulose acetate, a plastic substance that is slow to degrade in the natural environment, taking up to 18 months to biodegrade under normal conditions.(5,6) In addition to their persistence and prevalence in the environment, cigarette filters have been shown to leach toxic compounds from the discarded filter.(7,8) During the growing and production of tobacco cigarettes, various chemicals and additives are utilized, including pesticides, herbicides, fungicides, and insecticides.(9) Additionally, there are over 4000 chemicals present in tobacco smoke, including polycyclic aromatic hydrocarbons (PAHs), benzene, formaldehyde, aromatic amines, metals, etc.(10,11)
Previous studies have demonstrated chemicals in smoked cigarette leachate are acutely toxic to aquatic animals. Slaughter et al. observed an LC50 (lethal concentration that kills 50% of test specimens) of 1 cigarette butt/liter (CB/L) of water on saltwater topsmelt and freshwater fathead minnow test fish.(12) Earlier studies demonstrated a 48 h LC50 between 1 and 2 CB/L in Daphnia magna and a 48 h EC50 (a concentration at which 50% of specimens exhibit an effect) for immobilization of 0.06 CB/L in Ceriodaphnia dubia.(8,13) At a concentration of 5 CB/L, a 100% mortality rate was observed in three snail species after 8 days. Lower concentrations produced several behavioral changes.(14) Tobacco leachate affected the heart rate and disrupted the embryonic development of the medaka fish Oryzias latipes.(15) This was consistent with previous observations of a biphasic response to nicotine exposure.(16) More recently, it has been found that exposure to an aqueous cigarette tar extract resulted in reduced expression of cortical neural progenitor markers, decreased the number of cortical layer neurons, and caused a substantial loss of synaptic proteins in human embryonic stem cells.(17)
While biological end points such as mortality, development, and behavioral changes have been studied, there is little information on the specific biological pathways that smoked cigarette leachate may disrupt. One potential biological pathway is the aryl hydrocarbon receptor (AhR) pathway, which regulates a number of genes that encode xenobiotic metabolism enzymes as well as critical immunological and developmental pathways that are associated with carcinogenesis and developmental toxicity.(18)In vitro studies with mouse hepatoma cells found that cigarette smoke condensate transformed the AhR to an active transcription factor that played an important role in mediating the genotoxicity of this complex pollutant.(19) It has been also shown that tobacco extracts induced 7-ethoxyresorufin-O-deethylase (EROD) activity via activation of the AhR pathway in human cultured fibroblasts and keratinocytes,(20) suggesting the presence of AhR agonists in the tobacco cigarette.(21) In addition to the AhR pathway, cigarette smoke condensates were also found to bind to and transcriptionally activate estrogen receptor (ER) and to cause induction of ER-regulated genes.(22) While several studies have shown biological activity of extracts, none have identified the specific chemicals responsible for these activities.
In order to understand the potential impacts of smoked cigarette waste on aquatic environments and human health, it is important to assess the toxicity of smoked cigarettes and the biological pathways involved in their toxicity. This study aims to investigate different biological pathways in which smoked cigarette leachate (SCL) acts in vitro and determine individual chemicals in SCL responsible for the biological activities. Specifically, we will: (1) compare the toxicity and biological activities by freshwater and seawater SCL using a battery of cell-based assays, (2) investigate the biological activities in different fractions of SCL, and (3) identify individual chemicals responsible for one of the biological responses, AhR activity (Figure 1a). An effect directed analysis (EDA) approach coupled with nontargeted chemical analysis was used for AhR-active chemical identification (Figure 1b). EDA is an approach that relies on an observed biological response to narrow down potential toxic compounds, which has been successfully implemented to identify toxic compounds in environmental media, such as sediment and water samples.(23−25) We hypothesize that SCL exerts cytotoxicity and in vitro biological activity via disrupting multiple molecular pathways.

Figure 1

Figure 1. (a) Overall experimental design. SCL was tested in vitro for toxicity, estrogen receptor, AhR, and p53 response. SCL was then separated into fractions by polarity and retested in assays that exhibited a positive response during initial toxicological testing. Following testing of SCL fractions, the nontargeted chemical analysis was performed to identify compounds potentially responsible for biological response. (b) Steps of chemical data analysis. Nontargeted chemical analysis of SCL was conducted to isolate all compounds uniquely found in SCL fractions that exhibited biological response. All compounds were first screened for their absence in control samples and compiled into a preliminary list of potential compounds. Compounds from the preliminary list were then organized by the SCL fractions in which they were found. Compounds found only in fractions exhibiting biological response were then considered for confirmation with an authentic reference standard.

Materials and Methods

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Chemicals

17β-Estradiol (purity of 98%) and PCB126 (purity of 99%) were purchased from Sigma-Aldrich (St. Louis, MO). The p53 ligand (Mitomycin) was purchased from EMD Millipore Corp. (Billerica, MA). Solvents and other chemicals were of analytical grade and purchased from Fisher Scientific (Waltham, MA). Deionized water with electrical resistivity of 18.2 MΩ/cm was prepared using a Barnstead E-pure system. Nicotine-d4 and cotinine-d3 were purchased from Cambridge Isotope Laboratories Inc. (Tewksbury, MA), and a mixture of acenaphthene-d10, chrysene-d12, 1,4-dichlorobenzene-d4, naphthalene-d8, perylene-d12, and phenanthrene-d10 was purchased from Accustandard (New Haven, CT). 2-Methylindole (2-MI) was purchased from Combi-Blocks, Inc. (Combi-Blocks Inc., San Diego, CA).

Leachate Preparation

Smoked cigarettes were prepared at the University of California, San Francisco, using a TE10z smoking machine (Teague Enterprises, Woodland, California) operated in accordance with ISO Standard 3308:2000. Marlboro Red cigarettes (Phillip Morris, New York, New York) were ignited with a heated nickel-chromium wire on the first two puffs and puffed 10 times in total, resulting in an average cigarette weight of 0.39 g and ranging from 0.25–0.55 g/cigarette. Smoked cigarettes were extinguished by ejecting into a basin containing dry ice and stored at −20 °C until use.
Leachate was initially prepared by soaking smoked cigarettes in water for 24 h, at concentrations of 10 and 100 smoked cigarettes per liter of water (cig/L). Two batches of leachate were prepared for analysis. One batch was prepared with freshwater, while the second was prepared with seawater (34 ± 2 ppt). Freshwater was prepared with a 1:1 ratio of deionized water and dechlorinated tap water and aerated overnight. Seawater was obtained from Scripps Institute of Oceanography (La Jolla, CA) and held in a flow-through system with a 20 μm in-line fiber filter and a chiller unit.
Fifty smoked cigarettes were weighed (19.78 and 19.49 g for FW and SW leachate, respectively) using a Mettler Toledo XS105DU analytic balance (Mettler Toledo, Columbus, OH) and recorded. The 50 smoked cigarettes were then placed into 500 mL of water and mixed at a rate of 200 rpm for 24 h with an Arrow Engineering 6000 mixer (Arrow Engineering Inc., Rockford, IL). The leachate was then filtered into a vacuum flask through a 2.7 μm Whatman GF/D Glass Microfiber Filter (Whatman plc, Maidstone, United Kingdom) and stored in 500 mL amber vials at −20 °C. Cigarette-free freshwater and seawater controls were prepared using the same protocol without smoked cigarettes.

SPE Extraction and Fractionation for Bioassays

Solid-phase extraction (SPE) was used to separate compounds from the leachate mixture. First, 6 mL Oasis HLB cartridges (Waters Corporation, Milford, MA) were rinsed with 5 mL of dichloromethane (DCM), and then 5 mL of acetone. Next, the cartridges were conditioned with 5 mL methanol and 15 mL LC/MS grade water, respectively. After conditioning, cartridges were loaded with either 100 mL of sample or control. The flow rate was maintained at 1–2 drops per second. The cartridges were then washed with 5 mL of LC/MS grade water and vacuumed dried. Compounds were eluted from the cartridges using acetone, DCM, and hexane (4:3:2, v/v). The extracts were then blown to dryness with nitrogen gas and redissolved in 0.5 mL methanol. The extracts were stored at −20 °C before use.
For fractionation, each 200 mL of seawater or freshwater sample was divided into two 100 mL aliquots. The first 100 mL aliquot was passed through an SPE cartridge (Oasis HLB, 6 mL, 500 mg), eluted by 10%, 25%, 50%, 75%, and 100% MeOH/H2O in order, then evaporated to dryness with nitrogen gas, and redissolved in 0.5 mL of methanol, respectively. The second 100 mL of leachate was passed through a new SPE cartridge, eluted with 100% MeOH only, then evaporated to dryness with nitrogen gas, and redissolved in 0.5 mL of methanol. After SPE, the samples were serially diluted, and the highest concentrations of SPE-extracted leachate sample that showed no cytotoxicity were further assessed for biological activities as described below.

MTT Bioassay

Cytotoxicity of cigarette leachate samples was assessed by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay in three different indicator cell lines described below. In brief, 90 μL of assay medium at a density of 5 × 105 cells/mL was added to each well of the 96-well assay plate. Ten μL of positive controls (methanol and DMSO), negative controls (cell assay medium), and serially 2n times diluted samples of 10 cig/L, 100 cig/L, and SPE extractive at 100 cig/L were added to each well. The cells were incubated for 16 h at 37 °C, 5% CO2 in a humidified incubator. After incubation, 10 μL/well of 5 mg/mL MTT solution (Sigma cat. no. M5655) was added to the plate and incubated for 4 h. Then 50 μL/well of DMSO was added to the plate and shaken at room temperature for 15 min before reading at 595 and 650 nm.

ER, AhR, and p53 Activity Assays

The estrogenic receptor (ER), aryl hydrocarbon receptor (AhR), and p53 responses to cigarette leachate samples were assessed using the in vitro LUMI-CELL ER (BG1Luc4E2) assay, AhR assay (GeneBLAzer CYP1A1-bla LS-180, Life Technologies, Carlsbad, CA), and p53 assay (p53RE-bla HCT-116, Life Technologies, Carlsbad, CA). BG1Luc4E2 cells were provided by Dr. Michael Denison (University of California-Davis). GeneBLAzer CYP1A1-bla LS-180 and p53RE-bla HCT-116 cells were purchased from Life Technologies (Carlsbad, CA). ER, AhR, and p53 activities were expressed as 17β-estradiol equivalent (EEQ), aryl hydrocarbon toxicity equivalents (TEQ), and mitomycin equivalent (McQ), respectively.
BG1Luc4E2 cells were stably transfected with a human estrogen-responsive luciferase reporter gene plasmid (pGudLuc7ere) and selected using G418 resistance. BG1Luc4E2 cells were grown in RPMI 1640. The cells were transferred into flasks containing DMEM media (supplemented with 5% carbon stripped fetal calf serum and G418 sulfate solution) and incubated for 4 days before harvesting for BG1Luc4E2 bioassay plates. Cells were then plated in 96-well plates and incubated at 37 °C for 24 h prior to dosing. Then, the media solution in each well was removed, and 100 μL of DMEM containing the serially diluted samples to be tested was added to each well. The plate was incubated for 16 h before analysis of luciferase activity. After lysing the cells (Promega lysis buffer), the luciferase activity was measured in a GLOMAX Multi Detection System (Promega), with automatic injection of 50 μL of luciferase enzyme reagent (Promega) to each well. The relative light units (RLUs) measured were compared to that induced by the 17β-estradiol (E2) standard after subtraction of the background activity. For the AhR and p53 assay, CYP1A1-bla LS-180 and p53RE-bla HCT-116 cells were cultured and used to measure AhR and p53 activity following the manufacturer’s protocols. To calculate EEQs, TEQs and MCQ dose–response curves of the reference compounds E2, PCB126, and mitomycin were generated by SigmaPlot software (SPSS, Chicago, IL). Treatment and control in vitro bioactivity results from the laboratory exposures were examined using the student’s t test. Differences were considered significant at p < 0.05. All statistical analyses were conducted using PASW v.19 (SPSS, Chicago, IL).

Genotoxicity Assay

Each eluted fraction was resuspended in ultrapure water (1:5) and tested for genotoxicity in Salmonella typhimurium (strain TA 1535) by using the Moltox UMU Kit (ISO 13829:200 method). The bacterial strains contain the gene umuC fused to a lacZ reporter gene. When genetic damage is formed, the umuC gene is induced as part of the SOS repair–response system and the genotoxicity can be assessed by the simultaneous expression of the lacZ reporter gene, allowing indirect detection via the enzymatic activity of β-galactosidase.(26)
Genotoxicity was assessed by quantifying the induction ratio (IR) and the β-galactosidase activity of samples and controls (positive and negative) in relation to the growth factor (G) of the bacteria. First, the bacteria were cultured to the exponential phase at 37 °C for 2 h with shaking. Next, 10-fold dilutions of the exposed cultures were incubated for 2 h in TGA culture medium (30 μL of bacteria in 270 μL of TGA) at the same conditions in order to obtain the G by measuring the OD600 of each sample. Then, 30 μL aliquots of the samples were incubated at 28 °C for 30 min in 150 μL of B-buffer containing o-nitrophenol-β-galactopyranoside (ONPG substrate) in order to measure its conversion into o-nitrophenol at OD420. The assay was set up in 96-well plates, and each sample was tested at 1:1.5, 1:3, 1:6, and 1:12 dilutions with triplicates. An additional treatment with 30% S9 mix in the first incubation step was prepared to assess the transformation of any compound present in the sample into mutagenic metabolites through the cytochrome P450 MFO system. Positive controls were prepared using 2-aminoanthrace and 4-nitroquinoline-N-oxide (for S9 treatment). The sample was considered genotoxic when IR ≥ 1.5 and G ≥ 0.5.

Chemical Analysis of SPE Fractions

The SPE fractions were diluted 100 times (from a concentration of 10,000 cig/L to the original concentration of 100 cig/L) in DCM and then analyzed by Pegasus 4D comprehensive two-dimensional gas chromatography coupled to time-of-flight mass spectrometry (GC × GC/TOF-MS) (LECO, St. Joseph, MI). Instrument conditions used for analysis can be found in Table S1. LECO ChromaTOF software (version 4.50.8.0 optimized for Pegasus) was used for data processing. Automatic peak search was conducted using a signal-to-noise ratio of 10. In order to efficiently compare the chemical constituents of samples to controls, ChromaTOF’s add-in feature, “statistical compare”, was employed. Statistical compare is a post-processing software package that allows the user to compare different subsets of samples by automatically aligning chromatographic peak tables. Statistical compare bases peak table alignment on both the retention times and mass spectral similarity. The output from statistical compare was exported into Microsoft Excel (Microsoft Corporation, Redmond, WA) for further organization and analysis.
This analysis distinguished compounds found uniquely in sample groups from those present in the control group. As summarized in Figure 1b, all compounds found to be unique to the sample groups were consolidated into a single Excel file and organized by individual sample. As significant AhR response was only observed in the SPE-75%-MeOH-Series Fraction, SPE-100%-MeOH-Series Fraction, and SPE-100%-MeOH-Only Fraction for both freshwater and seawater, compounds found uniquely in those three sample groups were identified by statistical comparison as described above. Other fractions without AhR response were then manually reviewed for the presence of the suspected compounds. The final group of suspected compounds met the following criteria: (1) found only in SPE fractions with AhR response; (2) not found in any smoked cigarette free control samples; and (3) found in both freshwater SCL and seawater SCL. Compounds meeting all criteria were then grouped by level of confidence in the tentative identification based on the match to the 2014 National Institute of Standards and Technology’s (NIST) Mass Spectral Library. Compounds were considered identifiable if they met the following criteria: (1) similarity score ≥800 (the maximum similarity score is 999) and (2) intensity of the most prominent ions followed a similar rank order pattern to the NIST Library spectrum. Identifiable compounds were then confirmed using authentic standards. Compounds not meeting the above criteria were classified as “unknown”. If confirmed by authentic standards, compounds were then quantified in both freshwater and seawater SPE-100%-MeOH-Series Fractions using external calibration curves with internal standards.

Results

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Cytotoxicity and Cellular Activities of SCL

Cytotoxicity of serially diluted leachate samples (dilution factors are indicated as 2n, with dilution factor n = 2–64; e.g., SW1 indicated seawater sample was diluted 2 times, and FW3 indicated freshwater sample was diluted 8 times) was assessed using the MTT method. In AhR cells, no significant cytotoxicity was found in either seawater or freshwater leachate from smoked cigarettes at concentrations of 10 cig/L and 100 cig/L (Figure 2a,b). No cytotoxicity was found in SPE-extracted fresh water leachate samples (dilution factor of 2–16) in ER cells (Figure 2c). In contrast, dose-dependent toxicity was observed in SPE-extracted seawater leachate samples (dilution factor of 2–16) in ER cells (Figure 2c). The relative cell availability increased with dilution factor, and relative cell availability was over 90% in samples with the dilution factor of 16 (i.e., SW-SPE4, diluted 16 times; Figure 2c). Compared to ER cells, the same SW-SPE4 samples showed cytotoxicity in p53 cells (Figure 2d) as well as the FW-SPE4 samples (Figure 2d), suggesting that the p53 cells are more sensitive to the extracts than the ER cells. SPE5 and samples of higher dilution (>32 times dilution) had no observed toxicity (Figure 2d). Overall, 10 cig/L and 100 cig/L leachate samples showed negligible cytotoxicity, but some of the SPE-extracted samples were still cytotoxic with a dilution factor of 8. Generally, seawater samples were found to be more toxic than fresh water samples.

Figure 2

Figure 2. Cytotoxicity of 10 cig/L, 100 cig/L, and SPE-extracted 100 cig/L SCL samples in (a, b) AhR cells, (c) ER cells, and (d) p53 cells. Mean ± SD, n = 3. FW, freshwater; SW, seawater; and SPE, solid-phase extracted sample. Number n in the sample code indicates dilution of 2n times (dilution factors range from 2 to 64).

The highest concentrations of SPE-extracted leachate sample that showed no cytotoxicity in Figure 2 were further assessed for AhR, ER, and p53 activities. The SPE-extracted seawater leachate at 100 cig/L showed significantly higher ER activity than controls, averaging an EEQ of 85.0 pg/L (Figure 3a, Table 1). SW-100 cig/L, FW-100 cig/L, and SPE-extracted 100 cig/L leachate samples showed significantly higher AhR activities than the controls (Figure 3b), and the highest TEQ was found in SPE-extracted seawater 100 cig/L leachate samples (2.66 ng/L; Table 1). SW-100 cig/L, FW-100 cig/L, SPE-extracted 100 cig/L leachate samples showed significantly higher p53 activities than those of controls (Figure 3c; Table 1). To summarize, 10 cig/L leachate samples showed no significant ER, AhR, or p53 activities, while SPE-extracted seawater leachate samples at 100 cig/L had the highest EEQ, TEQ, and MCQ values. In accordance with the MTT results, seawater samples generally had higher biological responses than freshwater samples.

Figure 3

Figure 3. Bioactivities of 10 cig/L, 100 cig/L, and SPE-extracted 100 cig/L SCL samples. (a) RLU of E2 and samples, (b) blue/green ratio of PCB126 and samples, and (c) blue/green ratio of mitomycin and samples. The horizontal dotted line in (a–c) indicates the benchmark of activity. SW, seawater; FW, freshwater; 10cig, 10 cig/L sample; 100cig, 100 cig/L sample; and SPE, solid-phase extracted sample. Triangles represent seawater tests and circles represent freshwater tests. (d) The mean aryl hydrocarbon toxicity equivalents (TEQ) of fractionated (eluted by 10%, 25%, 50%, 75%, and 100% MeOH/H2O in order or 100% MeOH only) seawater and freshwater SCL samples (mean ± SD, n = 3).

Table 1. EEQ, TEQ, and MCQ of Leachate Samplesa
 EEQ (pg/L)TEQ (pg/L)MCQ (μg/L)
SWctrl<29.3<0.171.4 ± 0.2
SW10cig<29.3<0.172.1 ± 0.2
SW100cig<29.313.2 ± 0.6*26.1 ± 2.3*
SW100cigSPE85.0 ± 11.1*2,660 ± 109*41.9 ± 5.9*
FWctrl<29.3<0.17<1.7
FW10cig<29.310.1 ± 8.2<1.7
FW100cig<29.322.8 ± 0.5*21.1 ± 1.0*
FW100cigSPE<29.345.6 ± 0.3*30.3 ± 2.9*
a

Asterisk indicates a significant difference with control (p < 0.05); mean ± SD, n = 3.

Cytotoxicity, Genotoxicity, and AhR Activity of SCL Extract Fractions

Cytotoxicity to AhR cells was not observed in any samples during treatment. As for genotoxicity, the induction ratios(IR) of FW and SW samples treated with and without S9 are presented in Figure 4. The IRs of FW samples were all below 1.5, suggesting genotoxicity was negligible. In general, SW samples exhibited slightly higher IRs than those of FW samples, but their calculated growth factors were all lower than 0.5. These results indicated that genotoxicity assessed by UMU was not observed in the samples with a maximum concentration factor of 50, which may be due to the relatively lower sensitivity of the bacterium-based UMU assay compared to human cell-based assays. For AhR activity, the extracts eluted with the highest percentage of methanol induced the highest TEQs. Seawater samples (SW) consistently exhibited significantly higher TEQs than freshwater samples (FW). The highest TEQs were 3641 pg/L and 1362 pg/L for SPE-100%-MeOH-Series seawater samples and SPE-100%-MeOH-Series freshwater samples, respectively (Figure 3d).

Figure 4

Figure 4. Genotoxicity of fractionated SCL samples. IR was calculated as a quantitative measure of the genotoxicity of fractionated samples. For each sample dilution, β-galactosidase activity and growth factor were combined to form the IR. SW samples showed higher IRs than FW samples, but the growth factors were below 0.5 for all samples, suggesting their negligible genotoxicity. FW, freshwater; SW, seawater; SPE, solid-phase extracted sample. Mean ± SD, n = 3.

Chemical Analysis

Since SCL at a low concentration (10 cig/L) induced only AhR activity (Figure 3b), which raises high environmental concerns, chemical analysis was carried on the AhR-responsive samples. Isolation of compounds unique to AhR-responsive samples yielded 11 compounds that met the inclusion criteria (described in Materials and Methods). Of the 11 compounds, 5 were considered identifiable based on comparison to the matching library spectra (Table 2). Of the five identifiable compounds, three authentic standards could be obtained. The identity of 2-methylindole (2-MI, CAS 95-20-5) and diethyl phthalate (DEP, CAS 84-66-2) were confirmed; the other compound did not match the n-propylbenzamide in terms of GC retention time. Although the mass spectra matched, the n-propylbenzamide authentic standard first-dimension retention time was 2652.26 s (s), while the experimental compound had a first-dimension retention time of 3967.51 s. The other two tentatively identified compounds, 5H-1-pyridine (CAS 270-91-7) and cinnamic acid, β-[N-benzoylamino]-3,4-dihydroxy- (CAS 143815-48-9), were not confirmed due to the lack of commercial authentic standards. The mass spectra for each of the two identifiable compounds are shown in comparison to their respective mass spectral library hits in Figure 5. The remaining six compounds included in the final list of suspected compounds were classified as unknown, as they did not meet one or more of the required criteria to be considered identifiable. Their mass spectra are in the Supporting Information (Figure S1). Table S2 shows the mass spectra for the six unidentifiable compounds unique to the AhR responsive samples.

Figure 5

Figure 5. Peak true mass spectra of the five identifiable compounds and their matching mass spectra in the NIST database.

Table 2. Five Identified Compounds Unique to AhR-Responsive Samples
analyteCAS registry number1D, 2D retention timepeak area freshwaterpeak area saltwaterquant ionmolecular weightsimilarity scorelevel of identification
2-methylindole95-20-52298.96, 1.9342778010324130131.17878confirmed by authentic standard
5H-1-pyridine270-91-72057.6, 1.960477629847.8117117.15910strong mass spectra match
benzamide, N-propyl-10546-70-03967.51, 1.261237251248528105163.22972strong mass spectra match; not matched by authentic standard
cinnamic acid, β-[N-benzoylamino]-3,4-dihydroxy-143815-48-92739.71, 1.6043655.52984.3105299.28845strong mass spectra match
diethyl phthalate84-66-22781.68, 1.802165243226.2149222.24946confirmed by authentic standard

Quantification of 2-MI and AhR Activity Test

The average concentration of 2-MI was 12.03 μg/mL (0.09 mM) (n = 3) in the SPE-100%-MeOH-Only Fraction seawater samples and 12.41 μg/mL (n = 3) in the SPE-100%-MeOH-Only Fraction freshwater samples. Significant cytotoxicity of 2-MI was observed in AhR cells at a concentration of 40 mM (5250 μg/mL), and the LC50 determined using the MTT assay was 14 mM (1844 μg/mL) (Figure 6). 1.5 mM (197.55 μg/mL) 2-MI induced significant AhR activity with a TEQ of 82.7 ± 3.02 pg/L (Figure 6), suggesting 2-MI is a weak AhR agonist present in the SCL.

Figure 6

Figure 6. Cytotoxicity (left) and AhR activity (right) of 2-MI expressed as relative cell availability and blue/green ratio, respectively. Mean ± SD, n = 3.

Discussion

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We found that both seawater and freshwater leachate of smoked cigarettes exhibit induced significant AhR, ER, and p53 activities at 100 smoked cigarettes/liter and higher concentrations. However, there was no significant cytotoxicity and genotoxicity at these levels. Previous studies on the toxic constituents of tobacco products focused on the health effects of mainstream tobacco smoke (MSS) and sidestream tobacco smoke (SS) to humans.(27,28) Considering discarded smoked cigarettes as one of the most prevalent types of litter found in ocean beaches and inland waterways, it is critical to assess the environmental toxicity and risks to human health created by discarded smoked cigarettes. Once entering the aquatic environment, toxic chemicals can leach from discarded smoked cigarettes to fresh and seawater.(2) Previous toxicological studies focused on endpoints in aquatic invertebrates, and only a few studies have been performed in fish species.(12) While whole animal responses such as survival and behavior have been measured, the potential mechanisms and biological pathways of the acute toxicity of cigarette leachate are unclear. To our knowledge, the present study was the first to assess the cellular toxicity and activities of freshwater and seawater leachate of smoked cigarettes in vitro.
To identify the causative agent(s) of the biological activities of the SCL, the nontargeted chemical analysis was conducted on the leachate fractions having biological activities. Eleven compounds were identified. Of the 11 compounds, 2-MI and DEP were confirmed with authentic standards. 2-MI, an alkaline flavoring agent, has been previously identified in cut tobacco and particulate phase mainstream tobacco smoke.(29−31) 2-MI has been described as a weak AhR agonist in zebrafish,(32) but was not found to be a significant inducer of AhR activity in H4IIE-luc rat hepatoma cell lines or human HepG2 (40/6) cells.(33,34) However, 2-MI was reported to be a weak activator of AhR in human cell lines as well as a strong inhibitor of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) inducible expression of CYP1A1 mRNA.(35) In this study, we also found 2-MI as a weak AhR agonist, suggesting the presence of other AhR agonists in the SCL.
DEP is a plasticizer used in cosmetic products such as hair spray and nail polish.(36) DEP has been previously identified as a constituent in particulate phase mainstream tobacco smoke.(31) Additionally, DEP has been found in environmental matrices such as soil, surface water, and house dust.(37−39) It is listed by the United States Environmental Protection Agency (U.S. EPA) as a priority substance with endocrine disrupting action.(40) DEP was found to be a weak AhR activator in H4IIE-luc rat hepatoma cells.(41) DEP also induced a weak estrogenic response in the yeast screen with the approximate potency of 0.0000005 relative to 17β-estradiol(42) and showed a dose-dependent estrogenic effect in MCF-7 cells and CHO cells at a range of 1–100 μM.(43) The presence of DEP confirmed in seawater and freshwater SLC suggests DEP as one of the causative chemicals for both ER and AhR activities of SCL.
5H-1-Pyridine, tentatively identified in this study, has been found in MSS and in tobacco residue.(44,45) Thorough literature review yielded no results in terms of toxicological studies. As a result, the QSAR Toolbox v. 4.2 (www.qsartoolbox.org), developed by the Organization for Economic Co-operation and Development (Paris, France) and European Chemicals Agency (Helsinki, Finland), was used in an attempt to predict the AhR activity of 5H-1-pyridine. However, a lack of relevant data available to the QSAR Toolbox did not allow for a prediction.

Limitations and Implications

Seawater SCL extracts showed higher AhR responses than their corresponding freshwater extracts. We found one compound uniquely present in seawater extracts with AhR response and 14 compounds uniquely present in freshwater extracts with AhR response. As these compounds did not meet the inclusion condition of being found in both freshwater and seawater samples, they were not further investigated. However, it is possible that compounds unique to seawater samples enhance or stimulate the AhR response or that compounds unique to freshwater samples have an antagonistic effect. In addition, our study did not take into account compounds that were significantly more abundant in samples with biological response compared to samples with no biological response. These factors may limit our ability to completely identify individual compounds responsible for biological responses within our current study design. Furthermore, the chemical analysis conducted in this study is limited to GC amenable compounds such as volatile/semivolatile organic compounds.
With over 4000 compounds identified in tobacco smoke, assessing the toxicological contribution of individual tobacco constituents is highly challenging. One study investigating the contributions of individual constituents was able to attribute up to 40% of in vitro cytotoxicity caused by particulate phase MSS and up to 90% of in vitro cytotoxicity caused by gas vapor phase MSS to certain constituent classes; however, the remaining attribution to in vitro cytotoxicity was unable to be explained.(46) Furthermore, a 2016 study assessing the contributions of 8 heterocyclic aromatic amines to the overall bacterial mutagenicity of particulate phase MSS found that only 1% of the overall mutagenicity could be explained by the 8 constituents. Future research should investigate the difference between seawater SCL and freshwater SCL composition, since SPE extractive as well as fractionated seawater SCL exhibited a greater AhR response, while both seawater and freshwater controls showed negative in vitro. One potential explanation for this difference may be related to the difference in pH of seawater and freshwater. The alkaline conditions of seawater were suspected to influence the bioaccumulation of nicotine as well as DNA damage observed in marine worms, as nicotine is un-ionized and bioavailable in alkaline conditions.(47,48) The difference in the toxicity and composition of seawater SCL and freshwater SCL could have implications regarding the environmental risk assessment of littered smoked cigarettes, as different aquatic environments may leach different compounds from littered smoked cigarettes.

Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.9b00201.

  • Mass spectra of unknown compounds found in AhR-responsive samples; instrument conditions of GC × GC/TOF-MS for the chemical analyses; and chemical analysis information on six unknown compounds found in AhR-responsive samples (PDF)

Author Present Address

Department of Chemical Engineering, McGill University, Montréal, Quebec H3A 0C5, Canada

The authors declare no competing financial interest.

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Acknowledgments

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This project was funded by California Tobacco Related Disease Research Program (TRDRP: 23RT-0014H). We thank Adam Whitlatch for producing smoked cigarettes (cigarette butts), Kayo Watanabe for technical assistance on sample extraction and instrumentation, and Nautilus Environmental San Diego Laboratory for assistance on the generation of the smoked cigarette leachate.

References

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This article references 48 other publications.

  1. 1
    (2014) Global Tobacco: Key Findings Part 1 - Tobacco Overview, Cigarettes and the Future, Euromonitor International, London. http://www.euromonitor.com/global-tobacco-key-findings-part-1-tobacco-overview-cigarettes-and-the-future/report (accessed June 27, 2019).
  2. 2
    Novotny, T. E. and Slaughter, E. (2014) Tobacco product waste: an environmental approach to reduce tobacco consumption. Curr. Environ. Health. Rep. 1 (3), 208216, DOI: 10.1007/s40572-014-0016-x
  3. 3
    Munari, C., Corbau, C., Simeoni, U., and Mistri, M. (2016) Marine litter on Mediterranean shores: analysis of composition, spatial distribution and sources in north-western Adriatic beaches. Waste Manage. 49, 483490, DOI: 10.1016/j.wasman.2015.12.010
  4. 4
    (2017) International coastal cleanup 2017 report., Ocean Conservancy, Washington, DC https://oceanconservancy.org/wp-content/uploads/2017/06/International-Coastal-Cleanup_2017-Report.pdf (accessed June 27, 2019).
  5. 5
    Ach, A. (1993) Biodegradable plastics based on cellulose acetate. J. Macromol. Sci., Part A: Pure Appl.Chem. 30 (9–10), 733740, DOI: 10.1080/10601329308021259
  6. 6
    Brodof, T. A. (1996). The mechanisms of cellulose acetate degradation and their relationships to environmental weathering. In 50th Tobacco Chemists’ Research Conference, Richmond, VA.
  7. 7
    Hoffmann, D. H. I. (1997) The changing cigarette, 1950–1995. J. Toxicol. Environ. Health 50 (4), 307364, DOI: 10.1080/009841097160393
  8. 8
    Register, K. (2000) Cigarette Butts as Litter- Toxic as Well as Ugly?. Underwater Naturalist 25 (2), 2329
  9. 9
    Glantz, S. A., Slade, J., Bero, L. A., Hanauer, P., and Barnes, D. E. (1996) The Cigarette Papers,University of California Press, Berkeley, CA.
  10. 10
    Hoffmann, D., Hoffmann, I., and El-Bayoumy, K. (2001) The less harmful cigarette: a controversial issue. A tribute to Ernst L. Wynder. Chem. Res. Toxicol. 14 (7), 767790, DOI: 10.1021/tx000260u
  11. 11
    Li, S., Banyasz, J. L., Parrish, M. E., Lyons-Hart, J., and Shafer, K. H. (2002) Formaldehyde in the gas phase of mainstream cigarette smoke. J. Anal. Appl. Pyrolysis 65 (2), 137145, DOI: 10.1016/S0165-2370(01)00185-1
  12. 12
    Slaughter, E., Gersberg, R. M., Watanabe, K., Rudolph, J., Stransky, C., and Novotny, T. E. (2011) Toxicity of cigarette butts, and their chemical components, to marine and freshwater fish. Tob. control 20, i25i29, DOI: 10.1136/tc.2010.040170
  13. 13
    Warne, M. S. J., Patra, R. W., Cole, B., and Lunua, B. (2002). Toxicity and a Hazard Assessment of Cigarette Butts to Aquatic Organisms. Proceedings from The Royal Australian Society Chemical Institute, Australasian Society of Ecotoxicology and International Chemometrics Society, July, 2002, Sydney, Australia.
  14. 14
    Booth, D. J., Gribben, P., and Parkinson, K. (2015) Impact of cigarette butt leachate on tidepool snails. Mar. Pollut. Bull. 95 (1), 362364, DOI: 10.1016/j.marpolbul.2015.04.004
  15. 15
    Lee, W. and Lee, C. C. (2015) Developmental toxicity of cigarette butts-An underdeveloped issue. Ecotoxicol. Environ. Saf. 113, 362368, DOI: 10.1016/j.ecoenv.2014.12.018
  16. 16
    (1988) The Health Consequences of Smoking: Nicotine Addiction, A Report of the Surgeon General, U.S. Department of Health and Human Services, Rockville, MD. https://profiles.nlm.nih.gov/ps/access/nnbbzd.pdf (accessed June 27, 2019).
  17. 17
    Begum, A. N., Aguilar, J. S., and Hong, Y. (2017) Aqueous cigarette tar extracts disrupt corticogenesis from human embryonic stem cells in vitro. Environ. Res. 158, 194202, DOI: 10.1016/j.envres.2017.06.012
  18. 18
    Denison, M. S. and Nagy, S. R. (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol. 43 (1), 309334, DOI: 10.1146/annurev.pharmtox.43.100901.135828
  19. 19
    Dertinger, S. D., Silverstone, A. E., and Gasiewicz, T. A. (1998) Influence of aromatic hydrocarbon receptor-mediated events on the genotoxicity of cigarette smoke condensate. Carcinogenesis 19 (11), 20372042, DOI: 10.1093/carcin/19.11.2037
  20. 20
    Ono, Y., Torii, K., Fritsche, E., Shintani, Y., Nishida, E., Nakamura, M., Shirakata, Y., Haarmann-Stemmann, T., Abel, J., Krutmann, J., and Morita, A. (2013) Role of the aryl hydrocarbon receptor in tobacco smoke extract-induced matrix metalloproteinase-1 expression. Exp. Dermatol. 22 (5), 349353, DOI: 10.1111/exd.12148
  21. 21
    Kitamura, M. and Kasai, A. (2007) Cigarette smoke as a trigger for the dioxin receptor-mediated signaling pathway. Cancer Lett. 252 (2), 184194, DOI: 10.1016/j.canlet.2006.11.015
  22. 22
    Meek, M. D. and Finch, G. L. (1999) Diluted mainstream cigarette smoke condensates activate estrogen receptor and aryl hydrocarbon receptor-mediated gene transcription. Environ. Res. 80 (1), 917, DOI: 10.1006/enrs.1998.3872
  23. 23
    Bandow, N., Altenburger, R., Streck, G., and Brack, W. (2009) Effect-directed analysis of contaminated sediments with partition-based dosing using green algae cell multiplication inhibition. Environ. Sci. Technol. 43 (19), 73437349, DOI: 10.1021/es901351z
  24. 24
    Thomas, K. V., Hurst, M. R., Matthiessen, P., Sheahan, D., and Williams, R. J. (2001) Toxicity characterisation of organic contaminants in stormwaters from an agricultural headwater stream in south east England. Water Res. 35 (10), 24112416, DOI: 10.1016/S0043-1354(00)00535-2
  25. 25
    Thomas, K. V., Hurst, M. R., Matthiessen, P., and Waldock, M. J. (2001) Characterization of estrogenic compounds in water samples collected from United Kingdom estuaries. Environ. Toxicol. Chem. 20 (10), 21652170, DOI: 10.1002/etc.5620201005
  26. 26
    Oda, Y., Nakamura, S. I., Oki, I., Kato, T., and Shinagawa, H. (1985) Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens. Mutat. Res.-Environ. Muta. 147 (5), 219229, DOI: 10.1016/0165-1161(85)90062-7
  27. 27
    Johnson, J. D., Houchens, D. P., Kluwe, W. M., Craig, D. K., and Fisher, G. L. (1990) Effects of mainstream and environmental tobacco smoke on the immune system in animals and humans: a review. Crit. Rev. Toxicol. 20 (5), 369395, DOI: 10.3109/10408449009089870
  28. 28
    Andreoli, C., Gigante, D., and Nunziata, A. (2003) A review of in vitro methods to assess the biological activity of tobacco smoke with the aim of reducing the toxicity of smoke. Toxicol. In Vitro 17 (5–6), 587594, DOI: 10.1016/S0887-2333(03)00091-2
  29. 29
    (2012) Commission Implementing Regulation (EU) No 872/2012 of 1 October 2012 adopting the list of flavouring substances provided for by Regulation (EC) No 2232/96 of the European Parliament and of the Council, Introducing it in Annex I to Regulation (EC) No 1334/2008 of the European Parliament and of the Council and repealing Commission Regulation (EC) No 1565/2000 and Commission Decision 1999/217/EC Text with EEA relevance, European Commission, Brussels; https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32012R0872&from=EN, accessed 5/1/2018.
  30. 30
    Wang, X., Liu, S., Xia, Q., Zhao, G., Guo, J., and Xie, F. (2013) Trace analysis of alkaline flavors in cut tobacco by heart-cutting multidimensional GC-GC-MS. J. Sep. Sci. 36 (23), 37503757, DOI: 10.1002/jssc.201300836
  31. 31
    Moldoveanu, S. C. and St. Charles, F. K. (2007) Differences in the chemical composition of the particulate phase of inhaled and exhaled cigarette mainstream smoke. Beitr. Tab. Forsch. Int. 22 (4), 290302, DOI: 10.2478/cttr-2013-0834
  32. 32
    Brown, D. R., Clark, B. W., Garner, L. V., and Di Giulio, R. T. (2015) Zebrafish cardiotoxicity: the effects of CYP1A inhibition and AHR2 knockdown following exposure to weak aryl hydrocarbon receptor agonists. Environ. Sci. Pollut. Res. 22 (11), 83298338, DOI: 10.1007/s11356-014-3969-2
  33. 33
    Sovadinová, I., Bláha, L., Janošek, J., Hilscherová, K., Giesy, J. P., Jones, P. D., and Holoubek, I. (2006) Cytotoxicity and aryl hydrocarbon receptor-mediated activity of N-heterocyclic polycyclic aromatic hydrocarbons: Structure-activity relationships. Environ. Toxicol. Chem. 25 (5), 12911297, DOI: 10.1897/05-388R.1
  34. 34
    Hubbard, T. D., Murray, I. A., Bisson, W. H., Lahoti, T. S., Gowda, K., Amin, S. G., Patterson, A. D., and Perdew, G. H. (2015) Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles. Sci. Rep. 5, 12689, DOI: 10.1038/srep12689
  35. 35
    Stepankova, M., Bartonkova, I., Jiskrova, E., Vrzal, R., Mani, S., Kortagere, S., and Dvorak, Z. (2018) Methylindoles and methoxyindoles are agonists and antagonists of human aryl hydrocarbon receptor. Mol. Pharmacol. 93, 631644, DOI: 10.1124/mol.118.112151
  36. 36
    (2018) Phthalates, U.S. Food and Drug Administration, Silver Spring, MD. https://www.fda.gov/Cosmetics/ProductsIngredients/Ingredients/ucm128250.htm (accessed June 27, 2019).
  37. 37
    Liu, Q., Chen, D., Wu, J., Yin, G., Lin, Q., Zhang, M., and Hu, H. (2018) Determination of phthalate esters in soil using a quick, easy, cheap, effective, rugged, and safe method followed by GC-MS. J. Sep. Sci. 41 (8), 18121820, DOI: 10.1002/jssc.201701126
  38. 38
    Zhu, F., Mao, C., and Du, D. (2017) Time-resolved immunoassay based on magnetic particles for the detection of diethyl phthalate in environmental water samples. Sci. Total Environ. 601, 723731, DOI: 10.1016/j.scitotenv.2017.05.111
  39. 39
    Kadi, M. W., Ali, N., and Albar, H. M. S. A. (2018) Phthalates and polycyclic aromatic hydrocarbons (PAHs) in the indoor settled carpet dust of mosques, health risk assessment for public. Sci. Total Environ. 627, 134140, DOI: 10.1016/j.scitotenv.2018.01.146
  40. 40
    (2012). Endocrine Disruptor Screening Program (EDSP) Universe of Chemicals, U.S. Environmental Protection Agency, Washington, DC.
  41. 41
    Mankidy, R., Wiseman, S., Ma, H., and Giesy, J. P. (2013) Biological impact of phthalates. Toxicol. Lett. 217 (1), 5058, DOI: 10.1016/j.toxlet.2012.11.025
  42. 42
    Harris, C. A., Henttu, P., Parker, M. G., and Sumpter, J. P. (1997) The estrogenic activity of phthalate esters in vitro. Environ. Health Perspect. 105 (8), 802, DOI: 10.1289/ehp.97105802
  43. 43
    Kumar, N., Sharan, S., Srivastava, S., and Roy, P. (2014) Assessment of estrogenic potential of diethyl phthalate in female reproductive system involving both genomic and non-genomic actions. Reprod. Toxicol. 49, 1226, DOI: 10.1016/j.reprotox.2014.06.008
  44. 44
    Akalin, M. K. and Karagoz, S. (2011) Pyrolysis of tobacco residue: part 1. Thermal. BioResources 6 (2), 15201531
  45. 45
    Yin, C., Xu, Z., Shu, J., Wang, H., Li, Y., Sun, W., Zhou, Z., Chen, M., and Zhong, F. (2014) Study on the effect of potassium lactate additive on the combustion behavior and mainstream smoke of cigarettes. J. Therm. Anal. Calorim. 115 (2), 17331751, DOI: 10.1007/s10973-013-3478-4
  46. 46
    Stabbert, R., Dempsey, R., Diekmann, J., Euchenhofer, C., Hagemeister, T., Haussmann, H. J., Knorr, A., Mueller, B. P., Pospisil, P., Reininghaus, W., Roemer, E. (2017) Studies on the contributions of smoke constituents, individually and in mixtures, in a range of in vitro bioactivity assays. Toxicol. In Vitro 42, 222246, DOI: 10.1016/j.tiv.2017.04.003
  47. 47
    Wright, S. L., Rowe, D., Reid, M. J., Thomas, K. V., and Galloway, T. S. (2015) Bioaccumulation and biological effects of cigarette litter in marine worms. Sci. Rep. 5, 14119, DOI: 10.1038/srep14119
  48. 48
    Benowitz, N. L., Hukkanen, J., and Jacob, P. (2009). Nicotine chemistry, metabolism, kinetics and biomarkers. In Nicotine psychopharmacology, pp 2960, Springer, Berlin, Heidelberg.

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  • Abstract

    Figure 1

    Figure 1. (a) Overall experimental design. SCL was tested in vitro for toxicity, estrogen receptor, AhR, and p53 response. SCL was then separated into fractions by polarity and retested in assays that exhibited a positive response during initial toxicological testing. Following testing of SCL fractions, the nontargeted chemical analysis was performed to identify compounds potentially responsible for biological response. (b) Steps of chemical data analysis. Nontargeted chemical analysis of SCL was conducted to isolate all compounds uniquely found in SCL fractions that exhibited biological response. All compounds were first screened for their absence in control samples and compiled into a preliminary list of potential compounds. Compounds from the preliminary list were then organized by the SCL fractions in which they were found. Compounds found only in fractions exhibiting biological response were then considered for confirmation with an authentic reference standard.

    Figure 2

    Figure 2. Cytotoxicity of 10 cig/L, 100 cig/L, and SPE-extracted 100 cig/L SCL samples in (a, b) AhR cells, (c) ER cells, and (d) p53 cells. Mean ± SD, n = 3. FW, freshwater; SW, seawater; and SPE, solid-phase extracted sample. Number n in the sample code indicates dilution of 2n times (dilution factors range from 2 to 64).

    Figure 3

    Figure 3. Bioactivities of 10 cig/L, 100 cig/L, and SPE-extracted 100 cig/L SCL samples. (a) RLU of E2 and samples, (b) blue/green ratio of PCB126 and samples, and (c) blue/green ratio of mitomycin and samples. The horizontal dotted line in (a–c) indicates the benchmark of activity. SW, seawater; FW, freshwater; 10cig, 10 cig/L sample; 100cig, 100 cig/L sample; and SPE, solid-phase extracted sample. Triangles represent seawater tests and circles represent freshwater tests. (d) The mean aryl hydrocarbon toxicity equivalents (TEQ) of fractionated (eluted by 10%, 25%, 50%, 75%, and 100% MeOH/H2O in order or 100% MeOH only) seawater and freshwater SCL samples (mean ± SD, n = 3).

    Figure 4

    Figure 4. Genotoxicity of fractionated SCL samples. IR was calculated as a quantitative measure of the genotoxicity of fractionated samples. For each sample dilution, β-galactosidase activity and growth factor were combined to form the IR. SW samples showed higher IRs than FW samples, but the growth factors were below 0.5 for all samples, suggesting their negligible genotoxicity. FW, freshwater; SW, seawater; SPE, solid-phase extracted sample. Mean ± SD, n = 3.

    Figure 5

    Figure 5. Peak true mass spectra of the five identifiable compounds and their matching mass spectra in the NIST database.

    Figure 6

    Figure 6. Cytotoxicity (left) and AhR activity (right) of 2-MI expressed as relative cell availability and blue/green ratio, respectively. Mean ± SD, n = 3.

  • References

    ARTICLE SECTIONS
    Jump To

    This article references 48 other publications.

    1. 1
      (2014) Global Tobacco: Key Findings Part 1 - Tobacco Overview, Cigarettes and the Future, Euromonitor International, London. http://www.euromonitor.com/global-tobacco-key-findings-part-1-tobacco-overview-cigarettes-and-the-future/report (accessed June 27, 2019).
    2. 2
      Novotny, T. E. and Slaughter, E. (2014) Tobacco product waste: an environmental approach to reduce tobacco consumption. Curr. Environ. Health. Rep. 1 (3), 208216, DOI: 10.1007/s40572-014-0016-x
    3. 3
      Munari, C., Corbau, C., Simeoni, U., and Mistri, M. (2016) Marine litter on Mediterranean shores: analysis of composition, spatial distribution and sources in north-western Adriatic beaches. Waste Manage. 49, 483490, DOI: 10.1016/j.wasman.2015.12.010
    4. 4
      (2017) International coastal cleanup 2017 report., Ocean Conservancy, Washington, DC https://oceanconservancy.org/wp-content/uploads/2017/06/International-Coastal-Cleanup_2017-Report.pdf (accessed June 27, 2019).
    5. 5
      Ach, A. (1993) Biodegradable plastics based on cellulose acetate. J. Macromol. Sci., Part A: Pure Appl.Chem. 30 (9–10), 733740, DOI: 10.1080/10601329308021259
    6. 6
      Brodof, T. A. (1996). The mechanisms of cellulose acetate degradation and their relationships to environmental weathering. In 50th Tobacco Chemists’ Research Conference, Richmond, VA.
    7. 7
      Hoffmann, D. H. I. (1997) The changing cigarette, 1950–1995. J. Toxicol. Environ. Health 50 (4), 307364, DOI: 10.1080/009841097160393
    8. 8
      Register, K. (2000) Cigarette Butts as Litter- Toxic as Well as Ugly?. Underwater Naturalist 25 (2), 2329
    9. 9
      Glantz, S. A., Slade, J., Bero, L. A., Hanauer, P., and Barnes, D. E. (1996) The Cigarette Papers,University of California Press, Berkeley, CA.
    10. 10
      Hoffmann, D., Hoffmann, I., and El-Bayoumy, K. (2001) The less harmful cigarette: a controversial issue. A tribute to Ernst L. Wynder. Chem. Res. Toxicol. 14 (7), 767790, DOI: 10.1021/tx000260u
    11. 11
      Li, S., Banyasz, J. L., Parrish, M. E., Lyons-Hart, J., and Shafer, K. H. (2002) Formaldehyde in the gas phase of mainstream cigarette smoke. J. Anal. Appl. Pyrolysis 65 (2), 137145, DOI: 10.1016/S0165-2370(01)00185-1
    12. 12
      Slaughter, E., Gersberg, R. M., Watanabe, K., Rudolph, J., Stransky, C., and Novotny, T. E. (2011) Toxicity of cigarette butts, and their chemical components, to marine and freshwater fish. Tob. control 20, i25i29, DOI: 10.1136/tc.2010.040170
    13. 13
      Warne, M. S. J., Patra, R. W., Cole, B., and Lunua, B. (2002). Toxicity and a Hazard Assessment of Cigarette Butts to Aquatic Organisms. Proceedings from The Royal Australian Society Chemical Institute, Australasian Society of Ecotoxicology and International Chemometrics Society, July, 2002, Sydney, Australia.
    14. 14
      Booth, D. J., Gribben, P., and Parkinson, K. (2015) Impact of cigarette butt leachate on tidepool snails. Mar. Pollut. Bull. 95 (1), 362364, DOI: 10.1016/j.marpolbul.2015.04.004
    15. 15
      Lee, W. and Lee, C. C. (2015) Developmental toxicity of cigarette butts-An underdeveloped issue. Ecotoxicol. Environ. Saf. 113, 362368, DOI: 10.1016/j.ecoenv.2014.12.018
    16. 16
      (1988) The Health Consequences of Smoking: Nicotine Addiction, A Report of the Surgeon General, U.S. Department of Health and Human Services, Rockville, MD. https://profiles.nlm.nih.gov/ps/access/nnbbzd.pdf (accessed June 27, 2019).
    17. 17
      Begum, A. N., Aguilar, J. S., and Hong, Y. (2017) Aqueous cigarette tar extracts disrupt corticogenesis from human embryonic stem cells in vitro. Environ. Res. 158, 194202, DOI: 10.1016/j.envres.2017.06.012
    18. 18
      Denison, M. S. and Nagy, S. R. (2003) Activation of the aryl hydrocarbon receptor by structurally diverse exogenous and endogenous chemicals. Annu. Rev. Pharmacol. Toxicol. 43 (1), 309334, DOI: 10.1146/annurev.pharmtox.43.100901.135828
    19. 19
      Dertinger, S. D., Silverstone, A. E., and Gasiewicz, T. A. (1998) Influence of aromatic hydrocarbon receptor-mediated events on the genotoxicity of cigarette smoke condensate. Carcinogenesis 19 (11), 20372042, DOI: 10.1093/carcin/19.11.2037
    20. 20
      Ono, Y., Torii, K., Fritsche, E., Shintani, Y., Nishida, E., Nakamura, M., Shirakata, Y., Haarmann-Stemmann, T., Abel, J., Krutmann, J., and Morita, A. (2013) Role of the aryl hydrocarbon receptor in tobacco smoke extract-induced matrix metalloproteinase-1 expression. Exp. Dermatol. 22 (5), 349353, DOI: 10.1111/exd.12148
    21. 21
      Kitamura, M. and Kasai, A. (2007) Cigarette smoke as a trigger for the dioxin receptor-mediated signaling pathway. Cancer Lett. 252 (2), 184194, DOI: 10.1016/j.canlet.2006.11.015
    22. 22
      Meek, M. D. and Finch, G. L. (1999) Diluted mainstream cigarette smoke condensates activate estrogen receptor and aryl hydrocarbon receptor-mediated gene transcription. Environ. Res. 80 (1), 917, DOI: 10.1006/enrs.1998.3872
    23. 23
      Bandow, N., Altenburger, R., Streck, G., and Brack, W. (2009) Effect-directed analysis of contaminated sediments with partition-based dosing using green algae cell multiplication inhibition. Environ. Sci. Technol. 43 (19), 73437349, DOI: 10.1021/es901351z
    24. 24
      Thomas, K. V., Hurst, M. R., Matthiessen, P., Sheahan, D., and Williams, R. J. (2001) Toxicity characterisation of organic contaminants in stormwaters from an agricultural headwater stream in south east England. Water Res. 35 (10), 24112416, DOI: 10.1016/S0043-1354(00)00535-2
    25. 25
      Thomas, K. V., Hurst, M. R., Matthiessen, P., and Waldock, M. J. (2001) Characterization of estrogenic compounds in water samples collected from United Kingdom estuaries. Environ. Toxicol. Chem. 20 (10), 21652170, DOI: 10.1002/etc.5620201005
    26. 26
      Oda, Y., Nakamura, S. I., Oki, I., Kato, T., and Shinagawa, H. (1985) Evaluation of the new system (umu-test) for the detection of environmental mutagens and carcinogens. Mutat. Res.-Environ. Muta. 147 (5), 219229, DOI: 10.1016/0165-1161(85)90062-7
    27. 27
      Johnson, J. D., Houchens, D. P., Kluwe, W. M., Craig, D. K., and Fisher, G. L. (1990) Effects of mainstream and environmental tobacco smoke on the immune system in animals and humans: a review. Crit. Rev. Toxicol. 20 (5), 369395, DOI: 10.3109/10408449009089870
    28. 28
      Andreoli, C., Gigante, D., and Nunziata, A. (2003) A review of in vitro methods to assess the biological activity of tobacco smoke with the aim of reducing the toxicity of smoke. Toxicol. In Vitro 17 (5–6), 587594, DOI: 10.1016/S0887-2333(03)00091-2
    29. 29
      (2012) Commission Implementing Regulation (EU) No 872/2012 of 1 October 2012 adopting the list of flavouring substances provided for by Regulation (EC) No 2232/96 of the European Parliament and of the Council, Introducing it in Annex I to Regulation (EC) No 1334/2008 of the European Parliament and of the Council and repealing Commission Regulation (EC) No 1565/2000 and Commission Decision 1999/217/EC Text with EEA relevance, European Commission, Brussels; https://eur-lex.europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32012R0872&from=EN, accessed 5/1/2018.
    30. 30
      Wang, X., Liu, S., Xia, Q., Zhao, G., Guo, J., and Xie, F. (2013) Trace analysis of alkaline flavors in cut tobacco by heart-cutting multidimensional GC-GC-MS. J. Sep. Sci. 36 (23), 37503757, DOI: 10.1002/jssc.201300836
    31. 31
      Moldoveanu, S. C. and St. Charles, F. K. (2007) Differences in the chemical composition of the particulate phase of inhaled and exhaled cigarette mainstream smoke. Beitr. Tab. Forsch. Int. 22 (4), 290302, DOI: 10.2478/cttr-2013-0834
    32. 32
      Brown, D. R., Clark, B. W., Garner, L. V., and Di Giulio, R. T. (2015) Zebrafish cardiotoxicity: the effects of CYP1A inhibition and AHR2 knockdown following exposure to weak aryl hydrocarbon receptor agonists. Environ. Sci. Pollut. Res. 22 (11), 83298338, DOI: 10.1007/s11356-014-3969-2
    33. 33
      Sovadinová, I., Bláha, L., Janošek, J., Hilscherová, K., Giesy, J. P., Jones, P. D., and Holoubek, I. (2006) Cytotoxicity and aryl hydrocarbon receptor-mediated activity of N-heterocyclic polycyclic aromatic hydrocarbons: Structure-activity relationships. Environ. Toxicol. Chem. 25 (5), 12911297, DOI: 10.1897/05-388R.1
    34. 34
      Hubbard, T. D., Murray, I. A., Bisson, W. H., Lahoti, T. S., Gowda, K., Amin, S. G., Patterson, A. D., and Perdew, G. H. (2015) Adaptation of the human aryl hydrocarbon receptor to sense microbiota-derived indoles. Sci. Rep. 5, 12689, DOI: 10.1038/srep12689
    35. 35
      Stepankova, M., Bartonkova, I., Jiskrova, E., Vrzal, R., Mani, S., Kortagere, S., and Dvorak, Z. (2018) Methylindoles and methoxyindoles are agonists and antagonists of human aryl hydrocarbon receptor. Mol. Pharmacol. 93, 631644, DOI: 10.1124/mol.118.112151
    36. 36
      (2018) Phthalates, U.S. Food and Drug Administration, Silver Spring, MD. https://www.fda.gov/Cosmetics/ProductsIngredients/Ingredients/ucm128250.htm (accessed June 27, 2019).
    37. 37
      Liu, Q., Chen, D., Wu, J., Yin, G., Lin, Q., Zhang, M., and Hu, H. (2018) Determination of phthalate esters in soil using a quick, easy, cheap, effective, rugged, and safe method followed by GC-MS. J. Sep. Sci. 41 (8), 18121820, DOI: 10.1002/jssc.201701126
    38. 38
      Zhu, F., Mao, C., and Du, D. (2017) Time-resolved immunoassay based on magnetic particles for the detection of diethyl phthalate in environmental water samples. Sci. Total Environ. 601, 723731, DOI: 10.1016/j.scitotenv.2017.05.111
    39. 39
      Kadi, M. W., Ali, N., and Albar, H. M. S. A. (2018) Phthalates and polycyclic aromatic hydrocarbons (PAHs) in the indoor settled carpet dust of mosques, health risk assessment for public. Sci. Total Environ. 627, 134140, DOI: 10.1016/j.scitotenv.2018.01.146
    40. 40
      (2012). Endocrine Disruptor Screening Program (EDSP) Universe of Chemicals, U.S. Environmental Protection Agency, Washington, DC.
    41. 41
      Mankidy, R., Wiseman, S., Ma, H., and Giesy, J. P. (2013) Biological impact of phthalates. Toxicol. Lett. 217 (1), 5058, DOI: 10.1016/j.toxlet.2012.11.025
    42. 42
      Harris, C. A., Henttu, P., Parker, M. G., and Sumpter, J. P. (1997) The estrogenic activity of phthalate esters in vitro. Environ. Health Perspect. 105 (8), 802, DOI: 10.1289/ehp.97105802
    43. 43
      Kumar, N., Sharan, S., Srivastava, S., and Roy, P. (2014) Assessment of estrogenic potential of diethyl phthalate in female reproductive system involving both genomic and non-genomic actions. Reprod. Toxicol. 49, 1226, DOI: 10.1016/j.reprotox.2014.06.008
    44. 44
      Akalin, M. K. and Karagoz, S. (2011) Pyrolysis of tobacco residue: part 1. Thermal. BioResources 6 (2), 15201531
    45. 45
      Yin, C., Xu, Z., Shu, J., Wang, H., Li, Y., Sun, W., Zhou, Z., Chen, M., and Zhong, F. (2014) Study on the effect of potassium lactate additive on the combustion behavior and mainstream smoke of cigarettes. J. Therm. Anal. Calorim. 115 (2), 17331751, DOI: 10.1007/s10973-013-3478-4
    46. 46
      Stabbert, R., Dempsey, R., Diekmann, J., Euchenhofer, C., Hagemeister, T., Haussmann, H. J., Knorr, A., Mueller, B. P., Pospisil, P., Reininghaus, W., Roemer, E. (2017) Studies on the contributions of smoke constituents, individually and in mixtures, in a range of in vitro bioactivity assays. Toxicol. In Vitro 42, 222246, DOI: 10.1016/j.tiv.2017.04.003
    47. 47
      Wright, S. L., Rowe, D., Reid, M. J., Thomas, K. V., and Galloway, T. S. (2015) Bioaccumulation and biological effects of cigarette litter in marine worms. Sci. Rep. 5, 14119, DOI: 10.1038/srep14119
    48. 48
      Benowitz, N. L., Hukkanen, J., and Jacob, P. (2009). Nicotine chemistry, metabolism, kinetics and biomarkers. In Nicotine psychopharmacology, pp 2960, Springer, Berlin, Heidelberg.
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