Emerging investigator series: nontargeted screening of aryl hydrocarbon receptor agonists in endangered beluga whales from the St. Lawrence Estuary: beyond legacy contaminants

Abstract

The elevated concentrations of organohalogen contaminants in the endangered St. Lawrence Estuary (SLE) belugas have prompted the hypothesis that aryl hydrocarbon receptor (AhR) activity may be a contributor towards their potential adverse effects. While indirect associations between AhR and contaminant levels have been reported in SLE beluga tissues, AhR activity was never directly measured. Using bioassays and nontargeted analysis, this study contrasted AhR activity and agonist profiles between pooled tissue extracts of endangered SLE and non-threatened Arctic belugas. Tissue extracts of SLE belugas exhibited significantly higher overall AhR activity than that of Arctic belugas, with a 2000s SLE beluga liver extract exerting significantly higher activity than blubber extracts of SLE and Arctic belugas from the same time period. Contrary to our expectations, well-known AhR agonists detected by nontargeted analysis, including polychlorinated biphenyls (PCBs), were only minor contributors to the observed AhR activity. Instead, Tox21 suspect screening identified more polar chemicals, such as dyes and natural indoles, as potential contributors. Notably, the natural product bromoindole was selectively detected in SLE beluga liver at high abundance and was further confirmed as an AhR agonist. These findings highlighted the significance of the AhR-mediated toxicity pathway in belugas and underscored the importance of novel AhR agonists, particularly polar compounds, in its induction.

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Environmental significance

Aryl hydrocarbon receptor (AhR) induction has been identified as a potentially important pathway mediating the toxicity of chemical contaminants in the endangered St. Lawrence Estuary (SLE) belugas, but direct measurement of AhR activity has never been performed. Here, we measured AhR activity, identified AhR agonists in SLE belugas, and compared them with a non-threatened, less contaminated beluga population from the Canadian Arctic. The in vitro AhR activity of SLE belugas was significantly higher than that of Arctic belugas. Traditional halogenated AhR agonists contributed minorly to the AhR activity of SLE beluga tissues, while Tox21 suspect screening revealed polar chemicals as major agonists. Our findings underscore the significance of the AhR-mediated toxicity pathway in SLE belugas and highlight the importance of identifying novel AhR agonists in understanding the induction of AhR-mediated toxicity pathways.

1 Introduction

Despite recovery efforts, the population of approximately 1850 belugas inhabiting the St. Lawrence Estuary (SLE) in Canada is listed as endangered due to population decline.^1,2 Their lack of recovery may be partially attributed to the effects of high tissue levels of anthropogenic contaminants, given the SLE beluga habitat is proximal to highly industrialized and urbanized areas along the St. Lawrence River.^3,4 Indeed, elevated levels of halogenated contaminants including polychlorinated biphenyls (PCBs), organochlorine pesticides (OCs), polybrominated diphenyl ethers (PBDEs), and per- and polyfluoroalkyl substances (PFAS) have been reported in SLE belugas at some of the highest concentrations among marine wildlife species worldwide.^5–13 Elevated tissue levels of many halogenated contaminants have been linked to altered blubber lipid profiles and poor body condition in SLE belugas.^8,14,15 This has culminated in speculation that the SLE beluga contaminant burden may exert adverse effects.

Aryl hydrocarbon receptor (AhR) activity has been identified as a potentially significant toxicity pathway for chemical contaminants in SLE belugas, an idea which has been supported by the detection of elevated concentrations of the well-known AhR agonists PCBs in their blubber.¹⁰ Notably, a previous study reported the expression of the cytochrome P450 1A1 (CYP1A1) in various SLE beluga tissues, which is an indicator of AhR pathway induction,¹⁶ and concentrations of the halogenated flame retardant hexabromobenzene (HBB) in SLE beluga blubber have been positively correlated with Ahr gene transcription.¹⁷ Direct measurement of the AhR activity of Artic beluga blubber with a reporter assay has provided additional evidence for the induction of the AhR pathway in belugas.¹⁸ Distinct from belugas inhabiting the Arctic, carcass necropsies of SLE belugas indicated high incidences of parturition-associated complications (e.g., dystocia) and calf mortality.^19–24 It would therefore be of great interest to directly measure and compare the AhR activity in SLE and Arctic belugas, and identify the major AhR agonists driving toxicity.

While targeted chemical analysis and in vitro bioassays have commonly been used for the detection of traditional, non-polar AhR agonists (e.g., PCBs) in the environment,^25–27 the rise in nontargeted analysis has enabled the discovery of an even broader suite of novel AhR agonists.^28–32 Through the use of nontargeted screening tools, several recent studies have highlighted the presence of novel polar AhR agonists in sediment,^33,34 gulls,³⁵ and cetaceans³⁶ that were overlooked by traditional approaches. Interestingly, polar agonists, such as rutaecarpine, hydrocortisone, and alantolactone were found to contribute to nearly 50% of the AhR activity measured in the liver of a fin whale (Balaenoptera physalus) from Korea.³⁶ The United States Environmental Protection Agency's (U.S. EPA) Toxicology in the 21st Century (Tox21) program has spearheaded the creation of an accessible library of compounds known to exhibit bioactivity towards a suite of receptors, including AhR.^37–40 This has further provided an opportunity to systematically identify AhR agonists in SLE belugas beyond legacy contaminants.

We therefore aimed to combine the complementary techniques of nontargeted analysis and in vitro assays to systematically identify AhR agonists in SLE belugas. The goals of this study were to (1) directly measure the AhR activity of beluga tissue extracts using H4IIE-luc AhR bioassays, (2) conduct nontargeted identification of nonpolar halogenated compounds in beluga tissue extracts and evaluate their contributions to AhR activity, (3) identify polar AhR agonists in beluga tissue extracts using suspect screening against the Tox21 database, and (4) compare and contrast the AhR activity and agonist profiles of SLE and Arctic beluga tissues. This is the first study to systematically measure AhR activity and identify corresponding AhR agonists in SLE belugas.

2 Materials and methods

2.1 Chemicals and reagents

Silica gel (SiliCycle SilicaFlash P60) was obtained from Fisher Scientific (Ottawa, Ontario, Canada). Sulfuric acid was obtained from ACP Chemicals (Montreal, Quebec, Canada). 5-Bromoindole and 2,3,7,8-tetrachlorodibenzodioxin (TCDD) chemical standards were purchased from Toronto Research Chemicals (Toronto, Ontario, Canada). o-Aminoazotoluene was purchased from Sigma-Aldrich (St. Louis, Missouri, United States). Twenty native and seven mass-labeled PCBs (details in Table S1†) were purchased from Wellington Laboratories (Guelph, Ontario, Canada). Seven mass-labeled hydroxylated polybrominated and chlorinated diphenyl ethers (OH-BDEs and OH-Cl-BDEs, details in Table S2†) derivatives were purchased from AccuStandard Inc (New Haven, Connecticut, United States) or synthesized as described in a previous study with purities > 98%.⁴¹ 1037 ToxCast chemicals (provided in 96-well plates, 10–30 mM in dimethyl sulfoxide solution) were provided by the U.S. EPA through a material transfer agreement. Ultrapure water and methanol, high-performance liquid chromatography (HPLC) grade acetonitrile and hexanes, and American Chemical Society (ACS) grade dichloromethane (DCM) were obtained from Fisher Scientific (Ottawa, Ontario, Canada). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) cell viability assay kit was obtained from Biotium (Fremont, California, United States).

2.2 Sample collection and extraction

The overall experimental design for this study is outlined in Fig. S1.† Blubber and liver samples were collected from SLE belugas found dead during the spring-summer months between 1993–2017. Whale specimens included juvenile (1–7 years old) and adult (≥8 years old) females and males (see details in Table S3†). Samples were wrapped in solvent-rinsed aluminum foil and kept at −20 °C until analyses. Age was estimated by counting the annual growth layers in longitudinal tooth sections according to a previously established method.⁴² Sub-sampling of tissue was performed with stainless steel dissection scissors which were cleaned with methanol between samples. As a large quantity (∼10 g) of samples is essential for receptor activity detection, we decided to pool samples collected from similar time periods. To obtain a widespread and representative coverage of the chemicals that have impacted the SLE belugas over ∼three decades, three ∼10 g pooled SLE beluga tissue samples were prepared: one pool of 7 blubber samples from 1993–1998 (“1990s SLE Blubber”), one pool of 12 blubber samples from 2000–2017 (“2000s SLE Blubber”), and one pool of 15 liver samples from 2000–2017 (“2000s SLE Liver”). Because SLE beluga liver sampled prior to the year 2000 was not available for inclusion in the present study, blubber from the 1990s was prepared separately to allow for more direct comparisons between SLE liver and blubber sampled in the 2000s.

One ∼10 g pooled Arctic beluga sample was also prepared by pooling blubber from two male belugas (HI-14-06 37 years old, HI-14-11 24 years old) that were hunted offshore of Hendrickson Island near Tuktoyaktuk, Northwest Territories, Canada during the 2014 summer hunting season by local Inuvialuit hunters. These belugas were part of a population inhabiting the Eastern Beaufort Sea, which typically congregate in the Mackenzie River Estuary. Blubber from these whales was harvested and stored in solvent-rinsed glass screw-top containers at −20 °C prior to analysis.

Each of the four pooled beluga tissue samples were extracted and cleaned up with acetone:hexane (5 : 2, v/v) and gel permeation chromatography (GPC), respectively, using an adapted version of a previously described method.⁴³ Text S1 of the ESI† provides details regarding the sample extraction and clean up methods.

2.3 Aryl hydrocarbon receptor (AhR) bioassay screening

In this study, the H4IIE-luc cell line bioassay was used to evaluate AhR activities. The H4IIE-luc cell line was derived from a rat hepatoma cell line which was stably transfected with a firefly luciferase (luc) reporter construct. While this cell line was derived from rat hepatoma cells, evidence has supported the acceptability of cross-species extrapolation,⁴⁴ and it has been widely used to evaluate AhR activities of various marine mammal extracts and to estimate their potencies towards AhR across several species.^{35,36,45–47} We conducted sequence similarity analysis via the Sequence Alignment to Predict Across Species Susceptibility (SeqAPASS; U.S. EPA)⁴⁸ tool and determined the similarity between whole beluga AhR and whole rat AhR to be 69.68% (susceptibility cut-off 13.67%), and therefore this bioassay was deemed suitable for the purposes of this study.⁴⁴ Assays with H4IIE-luc cells were conducted as described previously^49,50 with minor modifications. The cells were inspected for cytotoxicity after exposure using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tetrazolium (MTT) cell assay as described previously.⁵¹ Details regarding the bioassays and AhR potency calculations are provided in Texts S2 and S3,† respectively.

2.4 Instrumental analysis via LC-Orbitrap

Ultrahigh-performance liquid chromatography coupled with Orbitrap high-resolution mass spectrometry operated under atmospheric-pressure chemical ionization (LC-APCI-Orbitrap) was used for the nontargeted screening of halogenated organic compounds that are poorly ionized under electrospray ionization (ESI). LC-ESI-Orbitrap was used for the suspect screening of Tox21 compounds which are polar and ionizable under ESI. Details for the LC-APCI-Orbitrap and LC-ESI-Orbitrap instrumental analysis methods are outlined in Texts S4 and S5,† respectively.

2.5 Quality assurance/quality control

The recovery of the sample extraction/cleanup method was evaluated using a mixture of seven mass-labeled hydroxylated polybrominated and chlorinated diphenyl ethers (OH-BDEs and OH-Cl-BDEs) and measured via LC-APCI-Orbitrap analysis. Recoveries ranged from 72 ± 5 % (3-OH-BDE-7) to 117 ± 18 % (6-OH-BDE-47) (see details in Table S2†), indicating the procedure's suitability for halogenated organic compounds. Alongside the batch of extracted pooled tissue samples, a procedural blank was prepared and used for blank subtraction (chemical analysis) or data normalization (bioassay analysis). For the LC-Orbitrap analysis, one procedural blank was incorporated along with each batch (n = 4) of tissue samples. Chemical standards were injected after each group of six sample injections to monitor the repeatability of the instrument. Methanol (ESI) or acetonitrile (APCI) injections were performed after each standard injection to monitor potential carry over.

2.6 Nontargeted analysis of halogenated compounds

Nontargeted data analysis was accomplished with a previously developed in-house R program.⁵² Details are provided in Text S6.† 90 peak features were detected as putative chlorinated or brominated compounds. The identities of select compounds, including bromoindole and PCBs, were confirmed using authentic analytical standards and were assigned confidence levels according to the Schymanski scale.⁵³ The finalized peak list was provided in Supplementary Data 1 of the ESI.†

In this study, PCBs were distinguished by their degree of chlorination rather than congener number, as specific PCB congeners could not be resolved using LC. This level of identification was considered acceptable since (1) specific PCB congener concentrations have already been extensively studied in SLE belugas, (2) our main objective for the nontargeted analysis was to qualitatively compare the relative organohalogen burdens across the four pooled beluga tissue extracts, and (3) our interest was primarily focused on identifying novel, rather than legacy, organohalogens in the SLE belugas.

2.7 Tox21 suspect screening

The experimental MS² spectra of each extracted LC-ESI-Orbitrap-MS/MS peak feature was matched to the in silico MS² spectra of the corresponding hit from a Tox21 database (created in-house, details provided in Text S7†), using the Competitive Fragmentation Modeling for Metabolite Identification version 4.0 (CFM-ID 4.0)⁵⁴ weighted dot-product calculation, and a score was generated. True compound identifications where at least one MS² fragment was matched returned a score > 0, while false positive hits without any matching MS² fragments returned a score of 0. In addition to in silico MS² matching, the ionization mode was predicted for each hit using the ‘rcdk’ R package, to increase the identification confidence. H-bond receptor and H-bond donor calculations were generated, and any hits detected in positive ion mode without an H-bond acceptor, and those detected in negative mode without an H-bond donor, were removed from the list of hits. Since many Tox21 compounds have been well studied, many of their experimental MS² spectra are available via the mzCloud database (https://www.mzcloud.org/). Therefore, to further improve confidence, we manually inspected experimental MS² spectra of each positive hit and compared them to experimental MS² spectra from mzCloud, where they were available. Since there is some overlap between Tox21 and ToxCast chemicals, we were able to verify select Tox21 chemicals (Table S5†) using authentic ToxCast analytical standards. Confidence levels (CLs) were assigned as follows: for confidence level 1 (CL = 1), all compounds were confirmed by authentic standards using retention time and MS² spectra. For level 2a (CL = 2a), compounds were verified by mzCloud MS² spectra and CFM-ID predicted MS². For level 2b (CL = 2b), compounds were verified by mzCloud MS² spectra only. For level 3 (CL = 3), compounds were verified by CFM-ID predicted MS² matching, but not mzCloud as their experimental MS² were not found in the mzCloud library. Lastly, level 4 (CL = 4) compounds were verified by CFM-ID predicted MS² but did not match the experimental MS² spectra found within the mzCloud library. Level 4 compounds were removed from the list of positive hits.

3 Results and discussion

3.1 AhR activity of beluga tissue extracts

Pooled tissue extracts of 1990s SLE blubber, 2000s SLE blubber, 2000s SLE liver, and 2014 Arctic blubber were tested for their activities towards the AhR receptor using an H4IIE-luc cellular bioassay at doses ranging from 0.02–0.3 g_tissue mL⁻¹ in the exposure media. All four extracts showed significant AhR agonistic activity (Fig. 1A). The lowest dose of 2000s SLE blubber to significantly activate AhR (% TCDD_max = 7.5 ± 0.4 %) was 0.15 g_tissue mL⁻¹, while the lowest dose of 2014 Arctic blubber to significantly activate AhR (% TCDD_max = 7.9 ± 0.3 %) was 0.3 g_tissue mL⁻¹. The higher AhR potency of 2000s SLE blubber compared to 2014 Arctic blubber may be reflective of differing contaminant-induced responses related to the distinct exposure histories of these two populations. Indeed, higher contaminant exposure of SLE belugas compared to Canadian Arctic belugas has been reported previously.^55–58 Further, high expression of CYP1A1, which is an indicator of AhR activity induction, has been reported in various tissues of belugas from both the SLE and Canadian Arctic.¹⁶ However, to the best of our knowledge, this is the first time that AhR activity has been measured for SLE belugas and directly compared to that of the Canadian Arctic population.

Fig. 1 AhR potencies of beluga tissue extracts. In vitro AhR screening revealed high AhR potency of 2000s SLE beluga liver, as well as higher AhR potency of 2000s SLE blubber compared to 2014 Arctic blubber. Red dashed line denotes the % TCDD_max of the sample preparation control (dose = 0.5%). Letters denote statistical significance. Note that the sample “1990s SLE Blubber” exhibited cytotoxicity in the dose range (Fig. S2†).

Out of all tested extracts, the 1990s SLE blubber demonstrated the highest AhR activity, and the lowest dose to significantly activate AhR (% TCDD_max = 7.04 ± 0.98 %) was 0.04 g_tissue mL⁻¹. The greater AhR activity of 1990s SLE blubber than 2000s SLE blubber was likely due to changing burdens of chemicals impacting the belugas over time. Indeed, decreasing temporal trends of chemicals such as PCBs (1987–2002),¹³ legacy PFAS (2000–2017),⁵⁹ and OCs (1987–2007)⁵ have been reported in SLE belugas. While the 1990s SLE blubber exhibited the highest AhR activity compared to the other tested samples, its potency appeared to decrease at doses greater than 0.04 g_tissue mL⁻¹. Consistent with this, an MTT cell viability assay (tested at a dose range of 0.04 to 0.6 g_tissue mL⁻¹) revealed that the 1990s SLE blubber induced cytotoxicity towards the H4IIE cells, while no such cytotoxicity was observed for the 2000s SLE blubber over the same dose range (Fig. S2†). This further supports the presence of a distinct array of cytotoxic chemicals in 1990s SLE blubber compared to 2000s SLE blubber.

For the three other tested samples (2000s SLE liver, 2000s SLE blubber, and 2014 Arctic blubber), the AhR response was dose-dependent, with % TCDD_max values for the maximum tested dose (0.3 g_tissue mL⁻¹) ranging from 7.87 ± 0.35 % (2014 Arctic blubber) to 15.6 ± 0.73 % (2000s SLE liver). This represents an order of activity across these samples of 2000s SLE liver ≫ 2000s SLE blubber > 2014 Arctic blubber. The higher induction of AhR potency by the 2000s SLE liver compared to blubber was unexpected: lipid-rich blubber tissue is typically considered the main reservoir for hydrophobic contaminants (e.g., PCBs and OCs) in mammals, which are expected to exhibit higher AhR potencies. These results suggest a higher burden of AhR agonists in beluga liver tissues than in blubber. Similar to our results, a previous study on marine mammals (i.e., harbor porpoise (Phocoena phocoena), harbor seal (Phoca vitulina), and Orca (Orcinus orca)) from the North and Baltic Seas reported AhR induction by liver extracts but not blubber.²⁷ Moreover, some recent studies have demonstrated the contributions of moderately polar and polar chemicals towards AhR activities in wildlife tissues.^35,36 Collectively, these results demonstrate that distinct AhR agonist profiles occur between various tissues of beluga, and between the blubber of the SLE and Arctic beluga populations.

3.2 Nontargeted analysis of organohalogens as potential AhR agonists

Many bioaccumulative organohalogen compounds (e.g., PCBs and OCs) are known to interact strongly with AhR.^60,61 Therefore, we opted to conduct nontargeted analysis of organohalogens in the beluga tissue extracts. In total, 35 unique organohalogen compounds were detected across all four of the beluga extracts. Overall, the 35 compounds were assigned to 8 structural classes (Fig. 2A): (1) “PCBs”, (2) “OH-PCBs”, (3) “MeSO₂-PCBs”, (4) “OH-MeSO₂-PCBs”, (5) “Organochlorine Pesticide-Related”, (6) “PBDE-Related”, (7) “Natural Products”, and (8) “Unknown”. The Unknown class consisted of peaks whose identities could not be confidently elucidated (see the list of compounds in Supplementary Data 1†). The PCBs (N = 6) and Organochlorine Pesticide-Related (N = 6) structural classes contained the most compounds, followed by Unknown (N = 5), MeSO₂-PCBs (N = 4) and Natural Products (N = 4) and PBDE-related (N = 4), OH-PCBs (N = 3), and OH-MeSO₂-PCBs (N = 2). The number of organohalogen compounds detected varied across the four beluga samples: 2000s SLE blubber contained the highest level of organohalogens (N = 35), followed by 2014 Arctic blubber (N = 22), 1990s SLE blubber (N = 16), and 2000s SLE liver (N = 8).

Fig. 2 (A) Heat map showing the 35 organohalogen compounds detected by nontargeted LC-APCI-Orbitrap. The compounds were assigned to 8 structural classes. White cells indicate <LOD. (B) Boxplots showing the relative abundances of organohalogen compound classes across the four beluga tissue extracts. Peak intensities have been normalized by the lipid mass of each sample.

3.2.1 Legacy organohalogens were not major AhR agonists in SLE beluga. Organochlorine pesticide-related compounds were found to be highly enriched in 1990s SLE blubber relative to both 2000s SLE blubber (∼100 times more abundant) and 2014 Arctic blubber (∼1000 times more abundant). Consistent with our findings, several organochlorine pesticides have previously been reported in the tissue of SLE belugas, including oxychlordane, nonachlor, perchloropentacyclodecane (Mirex), dichlorodiphenyltrichloroethane (DDT), and their related derivatives.^{8,11,12,62,63} Oxychlordane was the overall most abundant organohalogen detected across all beluga blubber samples (Fig. 3A), and was also the most highly enriched compound in 1990s SLE blubber relative to 2000s SLE blubber (∼200 times higher) and 2014 Arctic blubber (∼1600 times higher) (Fig. 3C). This was likely reflective of the strict regulations and phase-outs of organochlorine pesticides that were established in North America beginning in the 1970s, with chlordane in particular becoming restricted in the 1980s. Decreasing temporal trends of organochlorines have previously been reported in SLE beluga blubber from 1987–2002 (ref. 13) and 1987–2007.⁵

Fig. 3 Bubble plots displaying the abundance of organohalogen compounds detected via nontargeted LC-APCI-Orbitrap in (A) 1990s SLE blubber and (B) 2000s SLE blubber in terms of lipid-normalized peak intensity, and fold relative to 2014 Arctic blubber. Black coloured points represent points falling outside of the legend range. (C) Annotations for the top 10 most highly abundant organohalogens are shown in panels A and B.

A suite of PCBs and PCB metabolites was also detected in the beluga tissues. They were categorized by their chlorination number and their identities were supported by comparison to a mixture of chemical standards based on retention times (Fig. S5†) and characteristic isotopic peak patterns. The most abundant class in 2000s SLE blubber was MeSO₂-PCBs, which were ∼350 times more abundant in 2000s SLE blubber than 2014 Arctic blubber (Fig. 2B). The compound with the highest overall abundance in 2000s SLE blubber was a Cl₅ MeSO₂-PCB, which was ∼1100 times more abundant in 2000s SLE blubber than 2014 Arctic blubber (Fig. 3B and C). Other PCB metabolites, namely OH-PCBs and OH-MeSO₂-PCBs, were also more abundant (∼35 times and ∼10 times higher, respectively) in 2000s SLE blubber relative to 2014 Arctic blubber. Their parent compounds, PCBs, were similarly more abundant (∼70 times higher) in 2000s SLE blubber than 2014 Arctic blubber. A previous study comparing PCB burdens in SLE beluga and Canadian Arctic beluga brain, liver, and muscle also reported higher levels in SLE belugas,⁶³ suggesting that SLE belugas may be continuously exposed to PCBs. Interestingly, all four PCB-related classes (PCBs, OH-PCBs, MeSO₂-PCBs, and OH-MeSO₂-PCBs) were also found to be enriched in 2000s SLE blubber relative to 1990s SLE blubber (∼15, ∼2900, ∼5, and ∼3 times more abundant, respectively).

It has been well-established that some PCB congeners are potent AhR agonists.⁶⁴ However, the order of the PCBs and PCB metabolite loads across the beluga tissue samples in the present study (2000s SLE blubber > SLE 1990s blubber > 2000s SLE liver > 2014 Arctic blubber) was not consistent with the order of their AhR potencies, indicating that PCBs were unlikely to explain the majority of their measured AhR activities. To further investigate whether PCBs could be contributing to the AhR potencies, we subjected aliquots of beluga tissue extracts to sulfuric acid treatment (Text S8†). Sulfuric acid treatment can provide useful information about the types of contaminants present in the extracts, since legacy halogenated contaminants (e.g., dioxins and PCBs) are stable in sulfuric acid while other compounds are subject to degradation.⁶⁵ Pre- and post-acid treated extracts were then tested in the H4IIE-luc bioassay to compare their AhR potencies. Notably, acid treatment was found to significantly reduce the AhR potencies of 2000s SLE liver and 2000s SLE blubber (Fig. S6A and B†), suggesting that legacy halogenated contaminants were not major contributors to AhR activity induction in those tissues. Contrastingly, acid treatment had no significant impact on the AhR-mediated potency of the 1990s SLE blubber (Fig. S6C†). Notably, while a previous study reported the presence of some dioxin-like PCBs (e.g., PCB-126) in the blubber of SLE belugas found dead from 1987–1990, their levels corresponded to low TCDD-EQ_chem values ranging from 0.04–580 pg g⁻¹.¹¹ This confirmed that PCBs were not the major contributors towards AhR activity in SLE belugas. Additional work was done to confirm that polycyclic aromatic hydrocarbons (PAHs) were also not major contributors to AhR activity, as described in Text S10.† The calculated TCDD-EQ_chem for PAHs explained < 1% of the TCDD-EQ_bio of all four tissue extracts in the bioassay (Table S6†), suggesting that PAHs were only minor contributors to the observed AhR activity. This was similar to previous studies reporting relatively low contributions of PAHs towards AhR potencies of wildlife samples, such as in the liver of black-tailed gulls (Larus crassirostris) from Korea, wherein AhR-active PAHs were found to explain only 2.8–9.7% of the activity.³⁵ These results further supported that traditional nonpolar AhR agonists should not drive the AhR activity in SLE belugas.

3.2.2 Halogenated Natural Products were enriched in SLE belugas. Interestingly, the Natural Products class was detected exclusively in the 2000s SLE liver and blubber samples (<LOD in 2014 Arctic blubber and 1990s SLE blubber). Four compounds belonging to this class were detected in 2000s SLE blubber, with the most abundant being a compound tentatively assigned as a bromochlorobipyrrole-type natural product with the formula C₈H₂N₂Br₄Cl (m/z = 480.6603), based on the similarity of its predicted formula to natural halogenated bipyrroles known to be produced by marine bacteria.⁶⁶ Some previous studies have reported natural brominated and chlorinated methyl- and dimethyl bipyrroles in bottlenose dolphins (Tursiops truncatus) from Mexico and the United States.^67,68 Interestingly, the overall most highly abundant organohalogen in 2000s SLE liver (m/z = 193.9610, Fig. 4A) belonged to the Natural Products class. Based on its chemical formula, we suspected this compound to be a natural brominated indole (chemical formula: C₈H₆BrN). The presence of chlorinated (C₈H₆ClN) and iodinated (C₈H₆IN) indole analogues at lesser abundances (Fig. 4B) provided further support for this identification, as marine organisms have previously been reported to produce brominated-, chlorinated-, and iodinated-containing products together.⁶⁹ We then confirmed its identity using a 5-bromoindole chemical standard. While the specific bromine location(s) of the bromoindole(s) in 2000s SLE liver could not be elucidated, the retention times in the 2000s SLE liver and chemical standard matched, which supported 5-bromoindole as the possible structure (Fig. 4C). The concentration of the bromoindole in 2000s SLE liver was determined to be 80 ng g_tissue⁻¹. Brominated indoles have previously been reported in marine sediment and water extracts,⁷⁰ as well as in the blubber of various cetaceans found dead or captured in Mexico,⁷¹ but this marked the first detection of a brominated indole in belugas.

Fig. 4 (A) Bubble plot displaying organohalogen compounds detected via nontargeted LC-APCI-Orbitrap in 2000s SLE liver highlighted one highly abundant brominated compound with a m/z = 193.9610 (predicted chemical formula: C₈H₆BrN). (B) Chlorinated (C₈H₆ClN) and iodinated (C₈H₆IN) analogues were detected at lesser abundances. (C) The identity of bromoindole was confirmed by a chemical standard. (D) The AhR agonistic activity of 5-bromoindole was confirmed by a chemical standard. (E) Docking ligand-receptor simulation of beluga AhR/TCDD, and (F) beluga AhR/5-bromoindole.

3.3 Bromoindole identified as a novel AhR ligand unique to SLE beluga liver

Because of the high abundance of bromoindole(s) in the liver, and the high AhR-mediated potency of the 2000s SLE liver relative to the other tissue extracts, we suspected that this compound may be a contributor towards the 2000s SLE liver AhR activity. We therefore measured its activity in the H4IIE-luc bioassay using a 5-bromoindole chemical standard. Its AhR potency was verified in the bioassay, with the maximum tested dose (50 μM) producing a % TCDD_max of 25.4% (Fig. 4D), corresponding to a calculated TCDD relative potency (ReP) value of 1.2 × 10⁻⁵ (Text S3†).

Notably, the H4IIE-luc bioassay measures AhR potency towards rat AhR protein. To obtain a better understanding of the potential binding of bromoindole to beluga AhR (bAhR), we employed molecular docking analysis with the predicted crystal structure of bAhR (details described in Text S11†). We first performed such an analysis of bAhR with known ligand TCDD (Fig. 4E). The interaction between TCDD-bAhR was characterized as containing hydrogen bonds between the oxygen molecules of TCDD and three amino acid residues (i.e., HIS 290, THR 288, and GLN 382), and was further stabilized by hydrophobic interactions with several residues (i.e., THR 288, PHE 323, ILE 324, LEU 352, and ALA 380) and π-stacking between a TCDD aromatic ring and PHE 294. The estimated TCDD-bAhR binding energy was similar to that obtained by molecular docking analysis of TCDD with mouse AhR,⁷² which has 22 residues conserved with bAhR. Having thus gained confidence in the analysis of docking towards bAhR, we proceeded to molecularly dock 5-bromoindole (Fig. 4F). The 5-bromoindole-bAhR interaction contained one hydrogen bond between the pyrrole group of 5-bromoindole and the GLY 320 amino acid residue. The interaction was further stabilized by hydrophobic interactions with several residues (i.e., THR 288, PHE 294, PRO 296, PHE 323, PHE 350, and LEU 352) and π-stacking between the 5-bromoindole aromatic rings and HIS 290. The similar binding pose compared to TCDD indicates that 5-bromoindole interacts strongly with the bAhR binding pocket. Future studies are warranted to directly measure the binding to bAhR.

Following this, we conducted mass balance AhR potency analysis of 5-bromoindole. It was determined that the contribution (TCDD-EQ_chem/TCDD-EQ_bio × 100%) of bromoindole towards the AhR potency was 1.2% for 2000s SLE liver. As a more polar novel AhR ligand in SLE belugas compared to classic AhR agonists, this further supported our hypothesis that several polar compounds might drive the AhR activity in belugas.

3.4 Tox21 screening revealed polar AhR candidate agonists in SLE belugas

Recent studies have highlighted the potential importance of several novel moderately polar to polar AhR ligands in wildlife and sediment.^34,36 Indeed, in one study investigating AhR agonists in the liver of gulls from Korea, PAHs explained < 10% of total AhR-mediated potencies, while 15 polar known and novel AhR agonists explained 27–52%.³⁵ Inspired by this, we conducted suspect screening of polar Tox21 compounds by constructing an in silico Tox21 mass spectral database. Through this approach, we detected 43 putative AhR agonists (CL = 1–3; Fig. 5A) across the beluga tissue extracts (43 in 2000s SLE liver, 27 in 2000s SLE blubber, and 21 in 2014 Arctic blubber) (Table S5†) for which summed exposure-activity ratios (∑EARs; details described in Text S12†) were calculated (Fig. 5B). 15 total agonists with CLs of 1, 2a, and 2b (15 in 2000s SLE liver, 6 in 2000s SLE blubber, and 3 in 2014 Arctic blubber) were selected for further inclusion in a shortlist of AhR candidates.

Fig. 5 Tox21 screening of polar compounds detected via nontargeted LC-ESI-Orbitrap. (A) Confidence level definitions and the corresponding number of compounds within each confidence level detected across the three beluga extracts (2000s SLE liver, 2000s SLE blubber, and 2014 Arctic blubber). (B) The summed EAR of Tox21 AhR hits (CL = 1–3), showed alongside the measured AhR in vitro assay potency of the corresponding beluga tissue extracts. (C) The top 10 (by EAR) Tox21 AhR hits across the beluga tissue extracts (CL = 1–2b). (D) The chemical structures of select aromatic Tox21 AhR hits.

Strikingly, the relative EAR values across samples (2000s SLE liver ≫ 2000s SLE blubber > 2014 Arctic blubber) were in clear agreement with the in vitro bioassay responses at the maximum tested dose (2000s SLE liver ≫ 2000s SLE blubber > 2014 Arctic blubber) (Fig. 5B). The contributions of the top 10 AhR candidates towards the EARs are shown in Fig. 5C (details in Table S7†). Notably, the majority of candidates contained aromatic groups (Fig. 5D), which are known to be a key structural feature in many AhR agonists.³⁶ The candidate exhibiting the highest EAR (1.64 × 10⁵) in 2000s SLE liver was o-aminoazotoluene (m/z = 226.1337), a synthetic azo dye used in the colorization of oils, fats, and waxes, and in the production of pigments.⁷³ This compound was not detected in any of the beluga blubber extracts. Due to its high EAR value, we further confirmed the identify of o-aminoazotoluene via a purchased chemical standard (CL = 1, Fig. S7†). Notably, five additional compounds were categorized as being dye-related, and all six dye-related compounds were not detected in the 2014 Arctic beluga blubber. This may indicate a unique exposure of SLE belugas to dye production-related compounds. Indeed, the SLE habitat is located downstream from large cities and industrialized areas, and the use or manufacturing of dye-related products may explain this exposure. Interestingly, indole-3-acetic acid (m/z = 176.0703; CL = 2b) was also identified as an AhR candidate in 2000s SLE liver (EAR = 2.41 × 10⁴) and 2000s SLE blubber (EAR = 8.95 × 10²). Together with the detection of brominated indole, the presence indole-3-acetic acid demonstrates the likelihood for the SLE belugas to be exposed to a variety of natural indole derivatives, many of which may have been overlooked by the present study as they may not be included in the Tox21 database. Indeed, previous studies have demonstrated the AhR agonistic activity of a diverse array of indoles.^74,75

Other detected compounds were related to various categories including pesticides, fungicides, fragrances, pharmaceuticals, preservatives, and industrial manufacturing. A recent study reported the occurrence of industrial antioxidants and UV absorbents in the blubber and liver of SLE beluga carcasses from 2000–2017,⁷⁶ demonstrating the belugas' exposure to a wider variety of industrial-related chemicals that fall outside of the historically analyzed organohalogens. These results demonstrate the utility of Tox21 data for the screening and prioritization of known AhR-active compounds in wildlife and underscore that polar AhR candidates warrant more attention in future studies.

4 Implications

While it was long hypothesized that exposure to high levels of AhR agonists might have adverse impacts on SLE belugas, this hypothesis had never been tested with direct toxicity results. This study reported the first detection of greater AhR agonistic activity in SLE belugas than Arctic belugas, supporting the potential importance of AhR agonists. Surprisingly, the AhR activity could not be explained by the most well-studied AhR agonists (e.g., PCBs and PAHs). Instead, unknown and moderately polar, aromatic chemicals (e.g., dyes and indoles) accumulated in the liver appeared to be largely responsible for this activity.

Future research is needed to further confirm certain findings of this study. The minor contributions of nonpolar organohalogenated compounds towards AhR activity were supported by the selective toxicity of the 2000s SLE beluga liver extract and the decrease in toxicity following sulfuric acid treatment. However, additional studies are required to measure AhR-active dioxins, furans, and PCBs in SLE beluga extracts and further determine their exact contributions to the AhR activities. Due to the lack of available chemical standards for many chemicals detected by Tox21 suspect screening, we were not able to clearly pinpoint the chemicals driving the AhR-activity. Future work is warranted to employ nontargeted analysis and effect-directed analysis to identify the predominant AhR agonists in beluga tissues. Additionally, the H4IIE-luc AhR bioassay utilized in this study was created from rat cells. The same bioassay has been widely used in previous studies to identify AhR agonists in wildlife.^{35,36,45–47} Although sequence similarity analysis and molecular docking supported the cross-species extrapolation to beluga AhR, caution should be exerted when interpreting these results.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

The authors declare no conflicts of interest.

Acknowledgements

This research was supported primarily by the Fisheries and Oceans Canada's Whale Science for Tomorrow program and the Natural Sciences and Engineering Research Council of Canada (NSERC). The authors acknowledge the support of instrumentation grants from the Canada Foundation for Innovation, the Ontario Research Fund, and the NSERC Research Tools and Instrument Grant. We thank Lucky and James Pokiak for the provision of the Arctic beluga samples (scientific research License no. 2525 of the Aurora Research Institute) and the Tuktoyaktuk Hunter and Trappers Committee for the permission to perform bioassay-based investigations on those samples (License no. 5471). We thank the Department of Fisheries and Oceans, Maurice Lamontagne Institute (Mont-Joli, Québec, Canada) and all participants and partners of the stranding network Réseau québécois d'urgences pour les mammifères marins (RQUMM), including but not limited to Robert Michaud, Pierre Béland, Daniel Martineau, and Stéphane Lair, for the collection of samples from stranded belugas. We also thank Yves Morin and Véronique Lesage for age determination of the belugas.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4em00243a

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