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Published in final edited form as:
Environ Toxicol Chem. 2011 August ; 30(8): 1915–1925. doi:10.1002/etc.581.

Identification of Benzothiazole Derivatives and Polycyclic
Aromatic Hydrocarbons as Aryl Hydrocarbon Receptor Agonists
Present in Tire Extracts
Guochun He†, Bin Zhao†,‡, and Michael S. Denison†,*†Department of Environmental Toxicology, University of California, Davis, California, USA‡State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-

Environmental Sciences, Chinese Academy of Sciences, Beijing, China

Abstract
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Leachate from rubber tire material contains a complex mixture of chemicals previously shown to
produce toxic and biological effects in aquatic organisms. While the ability of these leachates to
induce Ah receptor (AhR)-dependent cytochrome P4501A1 expression in fish indicated thepresence of AhR active chemicals, the responsible chemical(s) and their direct interaction with the
AhR signaling pathway were not examined. Using a combination of AhR-based bioassays, wehave demonstrated the ability of tire extract to stimulate both AhR DNA binding and AhRdependent gene expression and confirmed that the responsible chemical(s) was metabolicallylabile. The application of CALUX (Chemical-Activated LUciferase gene eXpression) cellbioassay-driven toxicant identification evaluation not only revealed that tire extract contained avariety of known AhR-active polycyclic aromatic hydrocarbons, but also identified 2methylthiobenzothiazole and 2-mercaptobenzothiazole as AhR agonists. Analysis of a structurally
diverse series of benzothiazoles identified many that could directly stimulate AhR DNA binding
and transiently activate the AhR signaling pathway and identified benzothiazoles as a new class of
AhR agonists. In addition to these compounds, the relatively high AhR agonist activity of a large
number of fractions strongly suggests that tire extract contains a large number of physiochemicallydiverse AhR agonists whose identities and toxicological/biological significances are unknown.

Keywords

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Ah receptor; Tire; Benzothiazoles; CALUX; Toxicant identification evaluation

INTRODUCTION
Vulcanized tire rubber represents a complex composition of hydrocarbons, minerals, metals,
carbon blacks, process and extender oils and other substances that have undergone anextensive and complex series of processing steps. In addition to the principal ingredient ofrubber, tires contain a wide variety of chemicals that contribute to properties such as
softness and resistance to skid, rolling, abrasion, aging and others [1,2]. Chemical additives
included in the processing of tires to obtain these characteristics include highly aromatic oils
(known to contain high concentrations of polycyclic aromatic hydrocarbons [PAHs]),
metals, peroxides, benzothiazole (BT) derivatives, phenols, phthalates, aromatic amines, andother chemicals as well as those that are formed during the tire vulcanization process [1–3].
*

Corresponding Author: Michael S. Denison, Department of Environmental Toxicology, Meyer Hall, One Shields Avenue,
University of California, Davis, CA 95616, Tel: 530-752-3879, Fax: 530-752-3394, msdenison@ucdavis.edu.

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Many of these chemicals are known to be toxic and/or carcinogenic. Considering thenumber of tires currently used and those that are disposed of, the amount of tire particles onroadways released as a result of road wear, and the increasing and extensive use of shredded
or crumb tire in a wide variety of applications, a substantial amount of chemicals can and/or
are known to be released from vehicle tire rubber into the environment as a result ofweathering and leaching [1–4]. Accordingly, there are significant concerns regarding theenvironmental and toxicological impact of chemicals that can be released (leached) from car
tire rubber during weathering and numerous studies have examined the toxicity of tireleachate (reviewed in Wik and Dave [2]).

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Leachates or extracts of rubber tire have been shown to produce toxicity in a variety ofaquatic organisms, including fish, amphibians, invertebrates, bacteria and plants [1,2,5–9],
and in human lung cell lines [10]. While acute lethality was the most common effect,mutagenic, teratogenic, growth inhibition, oxidative stress and alterations in estrogen
receptor and progesterone receptor-dependent gene expression (i.e., endocrine disrupting
activity) have also been reported [11,12]. Water leachates of tires have been shown to
induce expression of cytochrome P4501A1 (CYP1A1) in fathead minnows (Pimephalies
promelas) [2] and rainbow trout (Onchorhynchus mykiss) [6]. Induction of CYP1A1 ismediated by the aryl hydrocarbon receptor (AhR), a ligand-dependent transcription factor
that mediates many of the toxic and biological effects of persistent organic pollutants such
as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals [13,14].
Together, these results suggest that a chemical(s) present in tire leachate is responsible foractivating the AhR and AhR-dependent gene (CYP1A1) expression, although the specificchemical(s) responsible for this activation was not identified.

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While the best studied and highest affinity ligands for the AhR are halogenated aromatichydrocarbons (HAHs), such as the polychlorinated dibenzo-p-dioxins (PCDDs),
dibenzofurans (PCDFs), and biphenyls (PCBs), and polycyclic aromatic hydrocarbons(PAHs), such as benzo[a]pyrene and dibenz[a,h]-anthracene, recent studies havedemonstrated that the AhR can be bound and activated by structurally diverse chemicals
with little similarity to the high affinity HAH and PAH ligands [13,15–19]. Although the
majority of these latter ligands are relatively weak agonists when compared to the very
potent HAHs and PAHs, their structural diversity clearly demonstrates the promiscuous
ligand-binding activity of the AhR and indicates that the overall inducing potency of a given
sample extract would likely result from the combined AhR stimulating activity of manydifferent chemicals present in the extract. This is supported by recent studies by Misaki andcoworkers [12], who demonstrated the presence of AhR agonist activity of organic extracts
from road dust (of which tire wear particles are a significant component). Their initial
toxicant-identification-evaluation (TIE) analysis using an AhR-based cell bioassay not only
suggested that PAHs and hydroxy-PAHs were likely involved in the induction response, but
indicated that a major contribution to the activity derived from unidentified highly polarchemicals, a unique observation since most AhR agonists are hydrophobic. However, itremains to be determined whether the AhR activity identified by these investigators asassociated with the road dust originated from tire wear particles or materials from othersources (i.e., asphalt, combustion particulates, etc.).
While the above studies clearly demonstrate the presence of AhR-active chemicals in tire
extracts and leachates, no responsible chemicals have yet been identified. In our previous
studies, we have utilized a combination of AhR-based bioassay systems to identify andcharacterize novel AhR agonists and antagonists [16,20–22] and demonstrated the presenceof AhR agonists in newspaper and inks [16,23]. We have expanded our studies to include
screening of extracts of diverse biological, environmental and commercial and consumer
products with the goal of identifying and characterizing novel AhR activators and/or

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inhibitors and to determine their AhR-dependent toxic and biological potency. Here we
described the results of studies using both in vitro and cell-based AhR bioassays coupledwith TIE approaches to isolate and identify AhR agonists present in organic solvent extractsof tire. These studies have led to the identification of various PAHs and several BT
derivatives as contributors to the overall AhR agonist activity of tire extract.

MATERIALS AND METHODS
Materials

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2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD, dioxin) was obtained from Steve Safe (Texas
A&M University, TX, USA), [γ32P]ATP (>5000 Ci/mmol) was purchased from Amersham(Piscataway, NJ, USA), dimethylsulfoxide (DMSO), hexane, toluene, ethyl acetate (EA),
and methanol (MeOH) were from Fisher Scientific (PA, USA) and poly dI dC was from
Roche Molecular Biochemicals (IN, USA). Cell culture reagents and media were purchasedfrom Gibco/BRL (NY, USA), G418 was from Gemini Bio-Products (CA, USA), andluciferase lysis and assays reagents from Promega (WI, USA). Truck tire tread was collectedfrom a highway roadside. Benzothiazole derivatives were purchased from several sources:
N-cyclohexyl-2-benzothiazolylsulfenamide, 2-(4-chlorophenyl)-benzothiazole, 2benzothiazolyl diethyldithiocarbamate, 2-(4-morpholinyldithio)-benzothiazole and (2benzothiazolylthio)acetic acid from TCI (OR, USA); 2-morpholinobenzothiazole and 2benzothiazolyl disulfide from Pfaltz and Bauer (CT, USA); N-t-butyl-2benzothiazolesulfenamide and N,N-dicyclohexyl-benzothiazolesulfenamide from Wako
(VA, USA); BT, 2-methylthiobenzothiazole (MTBT), 2-mercaptobenzothiazole (MBT), 2hydroxybenzothiazole (OBT), 2-(2-benzothiazolylthio)-ethanol, 2-benzothiazolamine and 2chloro-benzothiazole from Aldrich Chemical (MO, USA); 4-(2-benzothiazolyl)-2-methylbenzenamine, N-(2-benzothiazolylthio)-N-cyclohexyl-2-benzothiazolesulfenamide and 2,2'dithiobis (6-nitro-benzothiazole) from TimTec (DE, USA); 2-(benzylsulfinyl)-1,3benzothiazole from ChemBridge (CA, USA).
Tire sample preparation, fractionation and chemical analyses

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For analysis of total tire extract, 1 gram of washed inner tire tread was cut into small pieces
and immersed into 2 ml of DMSO overnight in a Teflon®-lined screw-cap tube, followed bycentrifugation for 15 min at 1500 rpm. The resulting supernatant (referred to as the originaltire extract) was transferred into a fresh tube and an aliquot was serially diluted (10–10,000fold range) in DMSO and 1 µl of each concentration analyzed for AhR agonist activity. For
fractionation studies, 16 g of tire tread was incubated in 100 ml of toluene overnight, thesupernatant collected and the tire fragments extracted twice more with 100 ml of toluene.
The combined toluene extract was evaporated to dryness, chromatographed through ahexane-washed silica gel column and the column sequentially eluted with 60 ml hexane, 60
ml hexane–toluene (1:1, v/v), 60 ml toluene and 60 ml MeOH to yield twenty-two primary
fractions of approximately 10 ml each. Fractions were dried and resuspended in 1 ml ofhexane and the AhR agonist activity in 1 µl of each fraction was determined using themouse hepatoma H1L1.1c2 cells bioassay as described below, with fractions 4 to 22
showing agonist activity. This concentration of hexane had no adverse effect on the cells(data not shown). The most active fraction (number 20) was subjected to a secondary
fractionation by silica gel column chromatography and eluted with 20 ml hexane–toluene(1:1, v/v), 20 ml toluene, 20 ml toluene–EA (1:1, v/v), 20 ml EA, and 20 ml MeOH to
obtain twenty secondary fractions of approximately 5 ml each. Fractions were dried andresuspended in 1 ml of hexane (fractions 1–17) or methanol (fractions 18–20) and agonist
activity in 1 µl of each fraction was determined, with positive AhR activity identified in
fractions 8 to 12 and 18 to 20. Hexane solvent blanks were also run before sample
fractionation to check for contaminants released from the chromatographic matrix. Analysis

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NIH-PA Author Manuscriptof these solvent blanks by gas chromatography-mass spectrometry (GC-MS) and gel
retardation analysis revealed no chemical contamination or AhR activity. Active fractions
were further analyzed by GC/MS in full scan mode using an electron impact ionization
mode on a HP5973 mass spectral detector tuned with perfluorotributylamine using thesystem auto-tune parameters. The mass spectrometer was interfaced with a HP 6890 gas
chromatograph equipped with a 30 m × 0.25 m, 0.25 µM DB-5ms phase column. Optimized
oven program used inlet, transfer line and quadrapole temperatures of 250°C, 280°C, and160°C, respectively. The oven program was an initial temperature of 50°C held for 1 min,ramped at 10°C/min to 320°C and held 10 min. Helium was used as the carrier gas at aconstant flow of 0.8 ml/min. Analyses were executed in the splitless mode.
Cell culture and induction of luciferase activity

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Analysis of chemicals, extracts and chromatographic fractions for their ability to stimulate
AhR-dependent gene expression was carried out using recombinant mouse hepatoma(Hepa1c1c7) cell-based CALUX (H1L1.1c2 and H1L6.1c2) and CAFLUX (H1G1.1c3)
clonal cell lines. The CALUX clonal cell lines are essentially identical and contain a stably
integrated AhR-/dioxin-responsive element (DRE)-driven firefly luciferase plasmid(pGudLuc1.1 or pGudLuc6.1, respectively) whose time course of luciferase induction varies
as a result of differences in intracellular localization and stability of the luciferase geneproduct (with H1L1.1c2 cells inducing rapidly and maximally by 4 to 6 h after agonist
treatment and H1L6.1 cells inducing more slowly with little activity at 4 to 6 h but withmaximal induction observed by 24 h after agonist treatment [20,21]). The CAFLUX clonalcell line contains a stably integrated AhR-/DRE-driven enhanced green fluorescent protein(EGFP) reporter gene plasmid whose activity can be repeated measured in intact cells fortime course studies [21,24]. Transcriptional activation in the CALUX and CAFLUX celllines occurs in a ligand-, dose- and AhR-dependent manner [20,21,24]. For CALUX
analysis, cells grown in white clear-bottomed 96-well microplates were incubated withcarrier solvent DMSO (10 µl/ml), TCDD (1 nM), the indicated concentration of total tire
extract, chromatographic fraction or the indicated compound for 4 h (in H1L1.1c2 cells) or
24 h (in H1L6.1c2 cells) at 37°C and luciferase activity measured in a Berthold microplate
luminometer as previously described [19–22]. For CAFLUX analysis, cells grown in black,clear-bottomed 96-well microplates were incubated with carrier solvent DMSO (10 µl/ml),
TCDD (1 nM) or the indicated concentration of total tire extract at 33°C for the indicatedtime. Concentrations of DMSO up to 2% produced no visual signs of cytotoxicity in
CALUX or CAFLUX cell lines. EGFP was measured in intact cells (without removal of
media) using a Fluostar microtiter plate fluorometer (Phenix Research Products) with an
excitation wavelength of 485 nm (25 nm bandwidth) and an emission wavelength of 515 nm(10 nm bandwidth) as previously described [24]. Normalization between experiments
involved adjusting the instrument fluorescence gain setting so that the level of EGFP
fluorescence induced by 1 nM TCDD resulted in 9,000 relative fluorescence units. Samples
were run in triplicate and the fluorescent activity present in wells containing media only
were subtracted from the fluorescence in all samples.
Preparation of cytosol and gel retardation analysis
Hepatic cytosol was prepared from male Hartley guinea pigs (250–300 g) in buffer
containing 25 mM Hepes (pH 7.5), 1 mM ethylenediamine tetraacetic acid, 1 mM
dithiothreitol, and 10% (v/v) glycerol as previously described and aliquots stored at −80°C[25]. Incubation of cytosol with TCDD or the indicated extracts or chemicals andsubsequent gel retardation analysis was carried out as described in detail [25]. Briefly, acomplementary pair of synthetic oligonucleotides containing the DRE3 binding site for thetransformed AhR:Arnt complex (5'-GATCTGGCTCTTCTCAC-GCAACTCCG-3' and 5'GATCCGGAGTTGCGTGAGAAGAGCCA-3') were synthesized, purified, annealed, andEnviron Toxicol Chem. Author manuscript; available in PMC 2012 August 1.

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radiolabeled with [γ32P]ATP. An aliquot (125 µl) of liver cytosol (8 mg of protein/ml) was
incubated with DMSO (2.5 µl), TCDD in DMSO (20 nM final concentration), 1.25 µl of theoriginal tire extract in DMSO or the indicated compound (200 µM each in DMSO) for 2 h at
20°C. A 10 µl aliquot of each incubation was analyzed by gel retardation analysis, andprotein-DNA complexes were resolved by non-denaturing polyarylamide gel electrophoresis
and autoradiography of the dried gel. The amount of 32P-labeled DRE present in the induced
protein-DNA complex was determined using a molecular dynamics phosphoimager.

RESULTS
Tire extract induces AhR-dependent gene expression

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The presence of AhR agonists in leachates of tires have been suggested from several studies
in aquatic organisms [2,6,12]. In order to systematically identify the responsible chemical(s),we first needed to confirm the presence of AhR agonists in tire extracts. The indicatedconcentration of tire extract was incubated for four hours with recombinant mouse hepatoma(H1L1.1c2) cells, which contain a stably integrated AhR-responsive luciferase reporter gene[22,21]. These results demonstrated that tire extract induced luciferase in a concentrationdependent manner in the H1L1.1c2 cells (Fig. 1A), producing a response between 40 to170% of that maximally induced by TCDD. This induction response was back calculated toresult from a range of extract that was equivalent to 0.5 µg to 0.5 mg of original tire.
Superinduction of reporter gene activity by this extract was unexpected, but not surprising
given our previous observation of the ability of other sample solvent extracts to produce asynergistically enhanced induction response [26]. Similar concentration-dependent
induction/superinduction by this crude tire extract was also observed in recombinant AhRresponsive guinea pig, rat and human cell lines containing a stably transfected AhRresponsive reporter gene (Supplemental Data, Fig. S1) demonstrating that this induction
response was not unique to the mouse AhR and that this extract contained AhR agonists.
Maximal induction in these cell lines was approximately 210, 170, and 230% of that inducedby 1 nM TCDD, respectively. While the exact mechanism(s) responsible for the observed
superinduction response is not known, we have previously described several possible
mechanisms that may contribute to synergistic increases in AhR-dependent gene expression[26].

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To determine whether the induction response was by metabolically stable HAHs that might
have been extracted from the tire and/or by other metabolically labile AhR ligands (such as
PAHs and others), we examined the time course of induction of AhR-dependent gene
expression in recombinant rat hepatoma (H1G1.1c3) cells that contain a stably transfected
AhR-responsive EGFP reporter gene. A major advantage of these cells is that the same cells
can be analyzed repeatedly in time course studies since measurement of EGFP activity is
carried out using intact cells in culture media [21,24]. While EGFP was induced in aconcentration-dependent manner, the magnitude of gene induction by the tire extract
initially increased over 48 hours and then progressively decreased over time (Fig. 1B);
TCDD continued to induce EGFP to a stable maximal activity. These results not only
indicate that the responsible AhR agonists extracted from tire are metabolically labile andproduce only a transient induction response, they also confirm that the responsible chemicals
are not HAHs.
Tire extract stimulates AhR transformation and DNA binding in vitro
While the cell-based bioassays demonstrate induction of AhR-dependent reporter geneactivity by the tire extract, they do not directly demonstrate the ability of chemicals in thetire extract to bind to and activate the AhR. To confirm the ability of the tire extract todirectly activate the AhR, we examined its ability to stimulate AhR transformation and DNA

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binding in vitro. Incubation of guinea pig hepatic cytosol with crude tire extract andsubsequent gel retardation analysis revealed that the tire extract did indeed induce AhR
transformation and DNA binding (Fig. 1C). Phosphoimager quantitation revealed that theamount of AhR:ARNT:DRE complex formed by incubation with tire extract wascomparable to that produced by a maximally stimulating concentration of TCDD (Fig. 1D).
These results suggest that the ability of the tire extract to stimulate AhR-dependent gene
expression is due to the ability of chemicals present in the extract to bind to and stimulate
AhR transformation and DNA binding.
TIE identification of AhR-active compounds in tire extracts

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In order to identify the specific chemical(s) responsible for activating the AhR and AhR
signaling pathway, a toxicant identification evaluation approach was used. Tire extract was
fractionated by silica gel column chromatography and progressively eluted with a series ofsolvents (hexane, hexane/toluene (1:1, v/v), toluene and then methanol) into 22 fractions(Fig. 2A). Aliquots of each fraction were exchanged into hexane and AhR agonist activity ineach fraction determined using the recombinant mouse hepatoma (H1L1.1c2) cells.
Interestingly, 19 of 22 fractions exhibited AhR agonist activity with a range in overall
induction between 15 to 130% of that maximally induced by TCDD (Fig. 2B). The presenceof AhR agonist activity in so many fractions suggests the presence of a large number ofstructurally different AhR active compounds. To further separate, analyze and identify
responsible AhR agonists, the most active fraction (#20) from the first fractionation scheme
was subjected to additional silica gel chromatography and progressively eluted with adifferent series of solvents (hexane/toluene (1:1, v/v), toluene, toluene/ethyl acetate (1:1, v/v), ethyl acetate and methanol) into 20 fractions (Fig. 2A). Aliquots of each fraction were
exchanged into hexane (fractions 1–17) or methanol (fractions 18–20) and AhR agonist
activity of each determined using the H1L1.1c2 cells. Positive AhR agonist activity (definedas >10% of that of that maximally induced by TCDD) was contained in fractions 8 to 12 and
18 to 20 (Fig. 2C). Instrumental analysis by GC/MS was carried out on each active fraction
and comparison of mass spectra and retention times to authentic standards revealed that
active fraction 18 was shown to contain several BT derivatives (specifically BT, MTBT,
MBT, and OBT (Fig.3 and Supplemental Data, Fig. S2); a number of PAHs were alsotentatively identified in fraction 9, although some of these PAHs were also present in otherfractions (structures shown in Fig. 3). Many of these PAHs are known AhR agonists [15,27–
29] or would be expected to have AhR agonist activity. The lack of chemical contaminantsor AhR activity in the hexane solvent blanks run before extract fractionation confirmed that
the AhR activity was derived from the tire extract.
MBT and OBT are AhR agonists

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While instrumental analysis indicated the presence of several BT derivatives in active
fraction 18, they do not confirm that these compounds are AhR active. Accordingly, we
examined the ability of commercially obtained BT, MTBT, MBT, and OBT to stimulate
AhR-dependent gene expression in the mouse H1L1.1c2 cell line (Fig. 4A). Although thesefour BTs have similar structures (Fig. 3), only MBT and OBT induced AhR-dependent gene
expression and they were relatively weak inducers (as compared to TCDD); BT and MTBT
were inactive. While OBT was relatively efficacious and demonstrated a full concentrationdependent induction of luciferase activity (with an EC50 of ~4 µM), MBT was only a partial
AhR agonist, with a concentration of 100 µM MBT inducing luciferase to approximately
50% of that produced by a maximal inducing concentration of TCDD (Fig. 4A). To examine
the metabolic stability of MBT and OBT as AhR agonists, we examined the ability of 10µM of each compound to induce luciferase activity in two mouse hepatoma (Hepa1c1c7)based CALUX clonal cell lines whose time course of maximal luciferase gene induction isrelatively rapid (4–6 h for H1L1.1c2 cells) or slow (24 h for H1L6.1c3 cells) [21]. The lower
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relative magnitude of luciferase gene induction at 24 h as compared to 4 h, compare to thatinduced by 1 nM TCDD, indicates that these inducers are metabolically labile(Supplemental Data, Fig. S3), consistent with the transient induction response by the crude
tire extract [Fig. 1B]. To demonstrate that the identified BTs can bind to and activate theAhR, we examined their ability to stimulate AhR transformation and DNA binding by gel
retardation analysis. These analyses not only demonstrated that OBT and MBT could
stimulate AhR transformation and DNA binding to between 40 to 50% of that of TCDD andconfirmed them as AhR agonists, but interestingly revealed that BT and MTBT also
stimulated AhR transformation and DNA binding, albeit only to 15 to 20% of that produced
by TCDD (Fig. 4B). While these results would suggest that BT and MTBT are weak AhR
agonists, their inability to induce AhR-dependent gene expression may result from lower
potency/affinity, more rapid metabolism within the cell and/or other factors.
Activation of the AhR by BT derivatives

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The above analysis demonstrates the ability of the OBT and MBT to bind to and activate the
AhR and AhR-dependent gene expression. While a previous study identified a BT derivative
as an AhR agonist [30], the widespread use of these chemicals in many materials andpotential exposure to these compounds raises the question as to whether AhR agonist
activity is a common property of other BT derivatives. Accordingly, we examined the abilityof sixteen commercially available BT derivatives (Fig. 5) to stimulate AhR-dependent
luciferase gene expression (Fig. 6A) as well as AhR transformation and DNA binding by gel
retardation analysis (Fig. 6B). Although some compounds were inactive as activators of
AhR-dependent gene expression in H1L1.1c2 cells after 4 h of incubation (compounds 2, 4–
6, 11), several compounds (namely, compounds 3, 8–10, 13, 14) were moderately active,
inducing luciferase reporter gene activity to between 40 and 100% of that obtained with amaximally inducing concentration of TCDD. To examine the metabolic stability of thesenovel AhR agonists, we further examined their ability to induce luciferase gene expression
after 24 h incubation using mouse hepatoma (H1L6.1c2) cells. Similar to the results with
OBT and MBT, most of the BT derivatives induced significantly less luciferase activity at
24 h as compared to 4 h (Fig. 6A), indicating that they are metabolically labile AhR
agonists. Interestingly, compound 14 induced significantly more luciferase activity at 24 hthan at 4 h, suggesting a somewhat different mechanism of action for this compound and/or
its metabolism/conversion into a more potent AhR agonist. The ability of these BT
derivatives to stimulate guinea pig AhR transformation and DNA binding in vitro (Fig.
6B,C) were relatively consistent with the mouse cell induction for compounds 1, 3, 7 to 10,
12 to 14 (Figs. 6A upper panel, 6C), indicating that they can directly activate the AhR;
compound 11 was inactive in both assays. While compounds 15 and 16 induced reporter
gene activity to 20 to 30 % of that of TCDD, they stimulated little or no AhR transformation
or DNA binding (Fig. 6B,C). In contrast, compounds 2,4 to 6 were similar to BT and MTBTin that they could stimulate AhR DNA binding (to 10–60% of that of TCDD [Fig. 6B,C]),yet did not induce AhR-dependent gene expression (Fig. 6A). Previous studies haveidentified chemicals that can bind to and activate the AhR in vitro, but fail to induce AhRdependent gene expression in cells in culture [15,16,18], most likely the result of rapid
metabolic degradation of the chemicals within the cells.

DISCUSSION
Tire wear components are present throughout the environment and numerous studies have
not only catalogued the specific chemicals leached from these materials, but they have also
reported on the toxic and biological consequences of exposure to leachates and extracts ofthese materials on the aquatic environment and organisms [1–3]. Human exposure tochemicals leached from tires, shredded tires and tire wear material can occur by dermal

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exposure from the environmental sources and ingestion of contaminated materials as well as
inhalation of airborne particulate matter derived from tire wear material [2]. Evaluating theexposure and possible toxicity of tire-derived materials becomes more complex consideringthat weathering and aging of tire materials as well as heating of the tires as a result of
roadway friction likely results in the generation of other chemicals with significantly
different biological and toxicological effects and potencies. Accordingly, tire rubber
contains a wide variety of chemicals that can produce biological and toxicological effects
through many distinct mechanisms [1–4]. The current study has only focused on only one
class of bioactive compounds, those that can interact with and active the AhR and AhR
signaling pathway. The AhR bioassay-based TIE analysis of crude organic tire extract
described here revealed that most fractions contained substantial AhR agonist activity.
When we consider the range of elution solvents used to generate these fractions and the
documented promiscuity of AhR ligand structure [15–19,31], it is very likely that the tire
extract contains a large number of structurally and physiochemically diverse AhR agonists,
only a few of which were identified in this report. Interestingly, superinduction of AhRdependent luciferase reporter gene activity was observed with the crude tire extract in eachof the CALUX cell lines from four different species (Fig. 1A and Supplemental Data, Fig.
S1) indicating a commonality in the mechanism(s) of this enhancement of the induction
response among species and cell lines. The ability of chemicals and solvent extracts ofenvironmental samples (e.g., soil and sediment) to synergistically enhance the CALUX
induction response to a level greater than that maximally induced by TCDD has beenpreviously reported [26,32,33]. While the exact mechanisms of CALUX superinduction
have not been defined, several mechanisms have been proposed, including chemicaldependent inhibition of protein synthesis that increases the stability of the ligand:AhR
complex, decreases the level of a labile protein that represses AhR functionality and/or
enhancement of AhR transcriptional activity via additional cell signaling pathways [26,32].
Given that the CALUX superinduction response was observed only with the crude tire
extract and not with any of its fractions, it is more likely that the crude extract contains both
AhR agonists and a chemical(s) that affects another cellular signal transduction pathway thataugments AhR-dependent luciferase expression from the reporter gene plasmid. Consistent
with this possibility are previous observations that chemical-dependent activation of theprotein kinase C signaling pathway or treatment of CALUX cells with selected
prostaglandins, known signaling factors, can synergistically enhance AhR-dependent gene
expression [26,32,33]. The mechanism and chemical(s) in the tire extract responsible for thesuperinduction response and the importance of this response on the overall toxicity of tire
extracts remain to be determined.

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Polycyclic aromatic hydrocarbons were found in numerous active fractions of tire extract
and many of the PAHs that were identified are either known to be AhR agonists or are
similar in structure to known AhR-active PAHs [15,27–29]. Accordingly, the PAHs likely
represent a significant contributor to the overall AhR agonist activity of crude tire extract.
Several BT derivatives were also specifically identified in tire extract and two of theidentified compounds (MBT and OBT) were confirmed to be AhR agonists by subsequent
analysis in rodent cell line-and guinea pig cytosolic AhR-based bioassays. Interestingly, it
was recently reported that injection of MBT into rainbow trout had no effect on AhRinducible CYP1A-dependent ethyoxyresorufin O-deethylase activity, suggesting that MBT
does not react with the AhR [7]. However, results of the present study clearly demonstratethat MBT is an AhR agonist in in vitro and cell-based AhR bioassays (Fig. 4). Similarly,
although BT was only a very weak AhR agonist in our mammalian cell bioassays (i.e., 100µM BT induced less than 10% of maximal AhR-dependent gene expression), a previous
study identified BT as a relatively potent AhR agonist of the human AhR expressed in yeast[30]. While the reasons for these differences in response remain to be determined, they very
likely result from species differences in the AhR in both assays (rodent versus guinea pig
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versus human) and/or differences in the overall rate of metabolism/degradation between
these experimental systems. In fact, although HAHs show similar rank order potency withregards to AhR ligand binding and AhR activation between species, dramatic differences inligand specificity and potency of nonHAH ligands between species (particularly between
human, rodent and fish AhRs) have been reported [31,34,35].

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The AhR bioassay screening of commercially available BT derivatives identified many
compounds with AhR agonist activity and significant diversity in response was observed
relative to the in vitro and cell-based bioassay systems. The ability of most of the BT
derivatives to stimulate AhR-dependent luciferase gene expression correlated reasonably
well with their ability to activate AhR transformation and DNA binding, consistent withthem being direct AhR ligands. Additionally, the relative potency of these compounds to
induce AhR-dependent gene expression was generally reduced with longer incubation times(compare 4 to 24 h of incubation in Fig. 6A), and this likely results from metabolic
degradation of the BT inducer. Interestingly, the greater degree of induction by 4(benzothiazolyl)-2-methyl-benzamine (compound 14) at 24 h (as compared to 4 h) suggested
that this compound was either metabolically stable (leading to increased AhR-dependent
gene expression for a longer period of time), metabolized into a more potent AhR agonist
and/or it exerted additional effects that enhanced luciferase reporter gene expression. This
compound has been previously shown to be a potent and selective antitumor agent andindependently identified as an AhR agonist [36]. N-(2-benzothiazolylthio)-N-cyclohexyl-2benzothiazolesulfenamide (compound 16) and 2,2'-dithiobis (6-nitro-benzothiazole)(compound 15) were novel AhR agonists in that they were active in the cell-based luciferase
assay yet inactive in stimulating AhR transformation and DNA binding in vitro. While theexact mechanism for this differential effect is not known, it is possible that these compounds
were metabolized into more potent AhR agonists in the intact cells (leading to AhRdependent gene induction), and their inactivity in the DNA binding assay resulted from thelack of conversion of the compounds into AhR agonists since this cytosolic-based assay
lacks major xenobiotic metabolizing enzymes. This observation is comparable to ourpreviously reported studies with bilirubin and biliverdin, which were very weak agonists in
the AhR DNA binding assay, yet were relatively potent inducers of AhR-dependent gene
expression in intact cells [37]. The stimulation of AhR transformation and DNA binding andlack of induction of AhR-dependent gene expression by the compounds 2-chlorobenzothiazole (compound 2), 2-benzothiazolyl diethyldithiocarbamate (compound 4), 2(benzylsulfinyl)-1,3-benzothiazole (compound 6) and (2-benzothiazolylthio)acetic acid(compound 5) are not surprising, given previously that have found many AhR agonists to besignificantly more potent in the in vitro, cell-free AhR bioassays as compared to cell-based
gene induction assays [15,16,18]. In the cell-free AhR transformation and DNA binding
bioassay, the agonist has direct access to the AhR in the cytosolic incubation and if it canbind to and activate the AhR, it will do so, resulting in a positive response. In contrast, for acompound to be positive in cell-based bioassays, it must enter the cell, avoiding
sequestration (by membranes, lipids, proteins, and organelles) and metabolism (bydegradation enzymes such as cytochrome P450s), and bind to the AhR, stimulating AhR
nuclear localization, transformation and DNA binding, and induction of gene expression, allwithin the time frame of the bioassay. Thus, positive activity in the cell bioassay isdependent upon a variety of conditions, factors and the physiochemical properties of theactivating chemical and those chemicals that do not induce AhR-dependent gene expression
are negatively affected by one or more of these aspects. Overall, the results of these studies
have identified BTs as a novel class of AhR agonists.
Benzothiazoles are a large class of widely-used and globally-synthesized industrial
chemicals that are used in a wide variety of applications, including vulcanization
accelerators in rubber production, as slimicides in the paper and pulp industry, fungicides,
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herbicides, antimicrobial and anti-algal agents and as corrosion inhibitors in cooling waterand in antifreeze for automobiles [2,38,39]. Considering that many of these uses result in
release of these chemicals into the environment, coupled with the considerable
environmental half-life of some BTs [38], it is not surprising that these compounds havebeen detected in water, soils, sediments, groundwater, municipal wastewater effluent,industrial emission, and atmospheric deposition [2,40]. Given the role of the AhR inmediating the toxic and biological effects of structurally diverse chemicals, BTs could
contribute to the overall impact and effect of environmental mixtures of AhR active
compounds. Additionally, given that numerous additives are used in the manufacture oftires, that AhR agonist activity was identified in almost all fractions of the tire solvent
extract and the documented promiscuity of AhR ligand structure [15,17,29,31], the overall
AhR agonist activity of tire extracts very likely results from a combination of many
distinctly different chemicals. The identification of MBT, OBT, and several commerciallyobtained BTs as novel AhR agonists provide us with more insight into the structure-activity
relationships that govern AhR agonist responses. Further studies are needed to assess thecontribution of other tire additives to the overall AhR agonist activity of tire extract and to
determine its potential for producing AhR-dependent toxic and biological effects.

Supplementary Material
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Refer to Web version on PubMed Central for supplementary material.

Acknowledgments
This work was supported by grants from the National Institutes of Environmental Health Sciences (R01ES07685
and Superfund Research Grant ES004699), the California Agricultural Experiment Station and the American
Taxpayers.

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Figure 1.

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Tire extract containS Ah receptor (AhR) agonist activity. (A) Concentration-dependent
induction of AhR-dependent luciferase reporter gene expression by crude tire extract in
H1L1.1c2 cells was determined after 4 hours of exposure as described in the Materials andMethods section. Values represent the mean ± standard deviation (SD) of triplicate
determinations and are expressed as a percent of the activity induced by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD). (B) Time course of induction of enhanced green
fluorescent protein (EGFP) gene expression by crude tire extract. H1G1.1c3 cells weretreated with TCDD (1 nM) or tire extract (equivalent to 0.005 mg of original tire) at 33°C
and EGFP fluorescence was measured at the indicated time points, as described in Materials
and Methods section. Values represent the mean ± SD of triplicate determinations. (C andD) Tire extract stimulates AhR transformation and DNA binding in vitro. Guinea pig hepatic
cytosol was incubated with 2.5 µl dimethylsulfoxide (DMSO), 20 nM TCDD (in DMSO), or
tire extract (equivalent to 0.625 mg of the original tire material) for 2 h at 20 °C. The
amount of AhR transformation/DNA binding was determined by gel retardation analysis (arepresentative gel is shown in C) and the amount of AhR:DNA complex quantified by
PhosphorImager analysis (D) as described in the Materials and Methods section. Values
represent the mean ± SD of at least triplicate determinations and are expressed relative to theamount of complex induced by TCDD.

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Figure 2.

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Ah receptor (AhR)-based chemically activated luciferase expression (CALUX) cellbioassay-guided toxicant identification evaluation of tire extract. (A) Tire tread was cut into
small pieces, extracted with toluene and fractionated using silica gel chromatography byelution with the indicated solvents to obtain twenty-two primary fractions. The strongly
inducing fraction 20 was subjected to a secondary fractionation using a silica gel column
chromatography and eluted with the indicated solvents to obtain twenty secondary fractions.
(B and C) The AhR agonist activity in each primary fraction was determined using themouse hepatoma H1L1.1c2 CALUX cell bioassay with (B) primary fractions 4 to 22 and(C) secondary fractions 8 to 12 and 18 to 20 containing AhR agonist activity.

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Figure 3.

Chemical structures of the benzothiazoles identified in fraction 18 and the polycyclicaromatic hydrocarbons tentatively identified in secondary fraction 9 of the tire extract.

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Figure 4.

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Ability of benzothiazoles identified in tire extract to stimulate Ah receptor (AhR)-dependent
gene expression and AhR DNA binding. (A) H1L1.1c2 cells were incubated with theindicated concentrations of benzothiazole (BT), 2-methylthiobenzothiazole (MTBT), 2mercaptobenzothiazole (MBT) or 2-hydroxyenzothiazole (OBT) at 37°C for 4 h and theluciferase activity determined as described in the Materials and Methods section. Values
represent the mean ± standard deviation (SD) of triplicate determinations and are expressed
as a percent of maximal luciferase activity induced by 1 nM 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD). (B) Guinea pig hepatic cytosol was incubated with carrier solvent (2.5 µl of
dimethylsulfoxide [DMSO]), 20 nM TCDD (in DMSO), or 200 µM of the indicatedbenzothizole compound for 2 h at 20 °C. Aliquots of each sample were analyzed by gel
retardation analysis as described in the legend to Figure 1. The amount of induced
AhR:DRE complex formed was expressed relative to that of TCDD, with values
representing the mean ± SD of at least triplicate determinations. DRE: dioxin responsive
element.

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Figure 5.

Chemical structures of commercially available benzothiazole derivatives analyzed for Ah
receptor (AhR) agonist activity.

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Figure 6.

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Benzothiazole (BT) derivatives induce Ah receptor (AhR)-dependent luciferase reporter
gene expression and stimulate AhR transformation and DNA binding. (A) Confluent plates
containing the recombinant mouse hepatoma cells were incubated with the indicatedbenzothiazoles (10 µM) for 4 h (H1L1.1c2 cells) or 24 h (H1L6.1c2 cells) at 37°C.
Dimethylsulfoxide (DMSO) (10 µl/ml final concentration) and 1 nM 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) were used as negative and positive controls. Luciferase
activity in cell lysates was determined as described in the Materials and Methods section.
Values represent the mean ± standard deviation (SD) of triplicate determinations and are
expressed as a percent of TCDD. (B and C) Guinea pig hepatic cytosol was incubated withcarrier solvent (2.5 µl of DMSO) or 20 nM TCDD (in DMSO), the indicated benzothiazoles(200 µM) for 2 h at 20 °C. Aliquots of each sample were analyzed by gel retardation
analysis as described in the legend to Figure 1. The amount of induced AhR:DRE complex
formed was expressed relative to that of TCDD, with values representing the mean ± SD of
at least triplicate determinations. DRE: dioxin responsive element.

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