Identification of benzothiazole derivatives and polycyclic aromatic hydrocarbons as aryl hydrocarbon receptor agonists present in tire extracts

Abstract

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 the presence 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, we have demonstrated the ability of tire extract to stimulate both AhR DNA binding and AhR-dependent gene expression and confirmed that the responsible chemical(s) was metabolically labile. The application of CALUX (Chemical-Activated LUciferase gene eXpression) cell bioassay-driven toxicant identification evaluation not only revealed that tire extract contained a variety of known AhR-active polycyclic aromatic hydrocarbons, but also identified 2-methylthiobenzothiazole 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 physiochemically diverse AhR agonists whose identities and toxicological/biological significances are unknown.

Common Commercial and Consumer Products Contain Activators of the Aryl Hydrocarbon (Dioxin) Receptor

“…The documented promiscuity of AhR ligands suggests that there actually exists a much larger spectrum of structurally diverse AhR ligands than has been currently identified. In fact, recent studies examining the ability of crude polar and nonpolar solvent extracts of a variety of environmental, biological, food and commercial and consumer products are consistent with this hypothesis [10]–[13]. To extend our previous studies demonstrating the presence of AhR agonists in crude extracts of newspapers and tires, we carried out preliminary screening analysis of DMSO extracts of a wide variety of commercial or consumer products using gel retardation analysis.…”

“…For AhR-dependent chemically activated luciferase expression (CALUX) analysis, recombinant guinea pig intestinal adenocarcinoma (G16L1.1c8), mouse hepatoma (H1L1.1c2), rat hepatoma (H4L1.1c4) and human hepatoma (HG2l1.1c3) cells, containing the stably integrated DRE-driven firefly luciferase reporter plasmid pGudLuc1.1, were grown, maintained, treated and luciferase activity measured as previously described in detail [13], [15]. Luciferase activity in each well was expressed relative to that induced by 1 nM TCDD.…”

“…The H1L1.1c2 cells are optimal for identifying AhR activators that are metabolically unstable and thus exhibit maximal induction within a few hours after addition to cells, while H1L6.1c2 cells are optimal for identifying AhR activators that are more metabolically stable and induce gene expression over a longer period of time [17]–[19]. For time-dependent analysis, cells grown in white clear-bottomed 96-well microplates were incubated with carrier solvent DMSO (10 µl/ml), TCDD (1 nM), or the indicated concentration of sample extract for 4 h (in H1L1.1c2 cells) or 24 h (in H1L6.1c2 cells) at 37°C and luciferase activity measured in a microplate luminometer as previously described [13]. For analysis of AhR-responsive endogenous CYP1A1 gene expression, mouse hepatoma (Hepa1c1c7) cells were treated with the indicated chemical/extract and mRNA for CYP1A1 and Rig-S15 (internal control for normalization using primers 5′-TTCCGCAAGTTCACCTACC and 5′-CGGGCCGGCCATGCTTTACG) amplified by RT-PCR as previously described in detail [14].…”

“…During our analysis of solvent extracts of food products for AhR agonists [10], the inadvertent use of a rubber cap liner instead of a Teflon cap liner on vials containing the extracting solvent (DMSO) revealed that chemicals readily extracted from the rubber cap liner could maximally activate AhR DNA binding; no activation was observed with DMSO stored in Teflon-capped vials. These results, coupled with our recent identification of AhR agonists in extracts of commercial newspapers [11], [12] and automobile tires [13], prompted the present investigation to determine how widely distributed AhR-active chemicals are in common commercial and consumer products (rubber, plastic, paper, etc.). Given the documented ability of the AhR to respond to a wide range of exogenous and endogenous chemicals, the present work not only contributes to our understanding of the diversity and widespread nature of AhR agonists, but identifies putative sources of AhR ligands that can complicate experimental studies of AhR signal transduction.…”

CARD9 impacts colitis by altering gut microbiota metabolism of tryptophan into aryl hydrocarbon receptor ligands

“…The H1L1.1c2 cell line containing a stably integrated dioxin response elements (DRE)-driven firefly luciferase reporter plasmid pGudLuc1.1 has been described previously 49,50 . The cells were seeded in 96-well plates at 7.5 × 10 4 cells/well in 100 μl of complete Dulbecco’s modified Eagle’s medium (DMEM) (with 10% heat-inactivated FCS, 50 IU/ml penicillin, and 50 μg/ml streptomycin; Sigma-Aldrich) and cultured (37 °C, 10% CO 2 ) for 24 h before treatment.…”