Identification of Hydrocarbon Sulfonates as Previously Overlooked Transthyretin Ligands in the Environment.

Incidences of thyroid disease, which has long been hypothesized to be partially caused by exposure to thyroid hormone disrupting chemicals (TDCs), have rapidly increased in recent years. However, known TDCs can only explain a small portion (∼1%) of in vitro human transthyretin (hTTR) binding activities in environmental samples, indicating the existence of unknown hTTR ligands. In this study, we aimed to identify the major environmental hTTR ligands by employing protein Affinity Purification with Nontargeted Analysis (APNA). hTTR binding activities were detected in all 11 indoor dust and 9 out of 10 sewage sludge samples by the FITC-T4 displacement assay. By using APNA, 31 putative hTTR ligands were detected including perfluorooctanesulfonate (PFOS). Two of the most abundant ligands were identified as hydrocarbon surfactants (e.g., dodecyl benzenesulfonate). Moreover, another abundant ligand was surprisingly identified as a disulfonate fluorescent brightener, 4,4'-bis(2-sulfostyryl)biphenyl sodium (CBS). CBS was validated as a nM-affinity hTTR ligand with an IC50 of 345 nM. In total, hydrocarbon surfactants and fluorescent brighteners explain 1.92-17.0 and 5.74-54.3% of hTTR binding activities in dust and sludge samples, respectively, whereas PFOS only contributed <0.0001%. Our study revealed for the first time that hydrocarbon sulfonates are previously overlooked hTTR ligands in the environment.

Building the Environmental Chemical-Protein Interaction Network (eCPIN): An Exposome-Wide Strategy for Bioactive Chemical Contaminant Identification.

Although advancements in nontargeted analysis have made it possible to detect hundreds of chemical contaminants in a single run, the current environmental toxicology approaches lag behind, precluding the transition from analytical chemistry efforts to health risk assessment. We herein highlighted a recently developed "top-down" bioanalytical method, protein Affinity Purification with Nontargeted Analysis (APNA), to screen for bioactive chemical contaminants at the "exposome-wide" level. To achieve this, a tagged functional protein is employed as a "bait" to directly isolate bioactive chemical contaminants from environmental mixtures, which are further identified by nontargeted analysis. Advantages of this protein-guided approach, including the discovery of new bioactive ligands as well as new protein targets for known chemical contaminants, were highlighted by several case studies. Encouraged by these successful applications, we further proposed a framework, i.e., the environmental Chemical-Protein Interaction Network (eCPIN), to construct a complete map of the 7 billion binary interactions between all chemical contaminants (>350,000) and human proteins (∼20,000) via APNA. The eCPIN could be established in three stages through strategically prioritizing the ∼20,000 human proteins, such as focusing on the 48 nuclear receptors (e.g., thyroid hormone receptors) in the first stage. The eCPIN will provide an unprecedented throughput for screening bioactive chemical contaminants at the exposome-wide level and facilitate the identification of molecular initiating events at the proteome-wide level.

Disclosing Environmental Ligands of L-FABP and PPARγ: Should We Re-evaluate the Chemical Safety of Hydrocarbon Surfactants?

Chemical contaminants can cause adverse effects by binding to the liver-fatty acid binding protein (L-FABP) and peroxisome proliferator-activated nuclear receptor γ (PPARγ), which are vital in lipid metabolism. However, the presence of numerous compounds in the environment has hindered the identification of their ligands, and thus only a small portion have been discovered to date. In this study, protein Affinity Purification with Nontargeted Analysis (APNA) was employed to identify the ligands of L-FABP and PPARγ in indoor dust and sewage sludge. A total of 83 nonredundant features were pulled-out by His-tagged L-FABP as putative ligands, among which 13 were assigned as fatty acids and hydrocarbon surfactants. In contrast, only six features were isolated when His-tagged PPARγ LBD was used as the protein bait. The binding of hydrocarbon surfactants to L-FABP and PPARγ was confirmed using both recombinant proteins and reporter cells. These hydrocarbon surfactants, along with >50 homologues and isomers, were detected in dust and sludge at high concentrations. Fatty acids and hydrocarbon surfactants explained the majority of L-FABP (57.7 ± 32.9%) and PPARγ (66.0 ± 27.1%) activities in the sludge. This study revealed hydrocarbon surfactants as the predominant synthetic ligands of L-FABP and PPARγ, highlighting the importance of re-evaluating their chemical safety.

Thiol Reactome: A Nontargeted Strategy to Precisely Identify Thiol Reactive Drinking Water Disinfection Byproducts.

The precise identification of predominant toxic disinfection byproducts (DBPs) from disinfected water is a longstanding challenge. We propose a new acellular analytical strategy, the 'Thiol Reactome', to identify thiol-reactive DBPs by employing a thiol probe and nontargeted mass spectrometry (MS) analysis. Disinfected/oxidized water samples had reduced cellular oxidative stress responses of 46 ± 23% in Nrf2 reporter cells when preincubated with glutathione (GSH). This supports thiol-reactive DBPs as the predominant drivers of oxidative stress. This method was benchmarked using seven classes of DBPs including haloacetonitriles, which preferentially reacted with GSH via substitution or addition depending on the number of halogens present. The method was then applied to chemically disinfected/oxidized waters, and 181 tentative DBP-GSH reaction products were detected. The formulas of 24 high abundance DBP-GSH adducts were predicted, among which nitrogenous-DBPs (11) and unsaturated carbonyls (4) were the predominant compound classes. Two major unsaturated carbonyl-GSH adducts, GSH-acrolein and GSH-acrylic acid, were confirmed by their authentic standards. These two adducts were unexpectedly formed from larger native DBPs when reacting with GSH. This study demonstrated the "Thiol Reactome" as an effective acellular assay to precisely identify and broadly capture toxic DBPs from water mixtures.

Proteome-wide effects of naphthalene-derived secondary organic aerosol in BEAS-2B cells are caused by short-lived unsaturated carbonyls.

Exposure to air pollution causes adverse health outcomes, but the toxicity mechanisms remain unclear. Here, we investigated the dynamic toxicities of naphthalene-derived secondary organic aerosol (NSOA) in a human bronchial epithelial cell line (BEAS-2B) and identified the chemical components responsible for toxicities. The chemical composition of NSOA was found to vary with six simulated atmospheric aging conditions (C1-C6), as characterized by high-resolution mass spectrometry and ion mobility mass spectrometry. Global proteome profiling reveals dynamic evolution in toxicity: Stronger proteome-wide impacts were detected in fresh NSOA, but the effects declined along with atmospheric aging. While Nrf2-regulated proteins (e.g., NQO1) were significantly up-regulated, the majority (78 to 97%) of proteins from inflammation and other pathways were down-regulated by NSOA exposure (e.g., Rho GTPases). This pattern is distinct from the reactive oxygen species (ROS)-mediated toxicity pathway, and an alternative cysteine reaction pathway was revealed by the decreased abundance of proteins (e.g., MT1X) prone to posttranslational thiol modification. This pathway was further validated by observing decreased Nrf2 response in reporter cells, after preincubating NSOA with cysteine. Ethynyl-naphthalene probe was employed to confirm the alkylation of cellular proteome thiols on the proteome-wide level by fresh NSOA via in-gel fluorescence imaging. Nontarget analysis identified several unsaturated carbonyls, including naphthoquinones and hydroxylated naphthoquinones, as the toxic components responsible for cysteine reactivity. Our study provides insights into the dynamic toxicities of NSOA during atmospheric aging and identifies short-lived unsaturated carbonyls as the predominant toxic components at the posttranslational level.

Triclosan is the Predominant Antibacterial Compound in Ontario Sewage Sludge.

Sewage treatment plants (STPs) accumulate both antibiotic and nonantibiotic antimicrobial compounds that can select for antibiotic resistant bacteria. Herein, we aimed to identify the predominant antibacterial compounds impacting E. coli from Ontario sewage sludge consisting of thousands of unknown compounds. Among the 10 extracted sludge samples, 6 extracts exerted significant growth inhibition effects in E. coli. A total of 103 compounds were tentatively detected across the 10 sludge samples by suspect screening, among which the bacterial enoyl-ACP reductase (FabI) inhibitor triclocarban was detected at the highest abundance. A hypomorphic FabI knockdown E. coli strain was highly susceptible to the sludge extracts, confirming FabI inhibitors as the primary antibacterial compounds in the sludge. Protein affinity pulldown identified triclosan as the major ligand binding to a His-tagged FabI protein from the sludge, despite the higher abundance of triclocarban in the same samples. Effect-directed analysis was used to determine the contributions of triclosan to the observed antibacterial potencies. Antibacterial effects were only detected in F17 and F18 across 20 fractions, which was consistent with the elution of triclosan and triclocarban in the same two fractions. Further, potency mass balance analysis confirmed that triclosan explained the majority (58-113%) of inhibition effects from sludge extracts. This study highlighted triclosan as the predominant antibacterial compound in sewage sludge impacting E. coli despite the co-occurrence of numerous other antibiotics and nonantibiotics.

Ecological Role of 6OH-BDE47: Is It a Chemical Offense Molecule Mediated by Enoyl-ACP Reductases?

Although hydroxylated polybrominated diphenyl ethers (OH-BDEs) are among the most abundant natural organobromine compounds, the fundamental biological rationale for marine organisms to produce OH-BDEs remains elusive. Herein, we demonstrated that natural OH-BDEs exerted strong antibacterial activities against Escherichia coli by inhibiting enoyl-[acyl-carrier-protein] reductase (FabI), while anthropogenic OH-BDEs were inactive. Distinct from E. coli, OH-BDE-producing marine γ-proteobacteria including Marinomonas mediterranea MMB-1 (MMB-1) and Pseudoalteromonas luteoviolacea 2ta16 (Pl2ta16) exhibited resistance to 6OH-BDE47. An alternative enoyl-[acyl-carrier-protein] (ACP) reductase, FabV, was detected in all three OH-BDE-producing marine γ-proteobacteria. Thermal stability and protein affinity purification studies revealed that 6OH-BDE47 did not bind to recombinant or endogenous FabV of MMB-1 or Pl2ta16, demonstrating that FabV was the primary mechanism for OH-BDE-producing marine γ-proteobacteria to be resistant to 6OH-BDE47. To further confirm if the laboratory results were evidenced in the field, the 16S rRNA sequencing and metagenomics data from seven field-collected marine sponges were analyzed. Notably, the two Clade 4 sponges containing high concentrations of 6OH-BDE47 exhibited a distinct microbiome community structure compared to the other analyzed clades. Correspondingly, FabV was found to be selectively enriched in the same Clade 4 sponges. The merged evidence from the laboratory experiments and field studies demonstrated that 6OH-BDE47 may act as a chemical offense molecule in marine sponges.

Defining the Chemical Additives Driving In Vitro Toxicities of Plastics.

Increases in the global use of plastics have caused concerns regarding potential adverse effects on human health. Plastic products contain hundreds of potentially toxic chemical additives, yet the exact chemicals which drive toxicity currently remain unknown. In this study, we employed nontargeted analysis and in vitro bioassays to identify the toxicity drivers in plastics. A total of 56 chemical additives were tentatively identified in five commonly used plastic polymer pellets (i.e., PP, LDPE, HDPE, PET, and PVC) by employing suspect screening and nontargeted analysis. Phthalates and organophosphates were found to be dominant in PVC pellets. Triphenyl phosphate and 2-ethylhexyl diphenyl phosphate accounted for a high amount (53.6%) of the inhibition effect of PVC pellet extract on human carboxylesterase 1 (hCES1) activity. Inspired by the high abundances of chemical additives in PVC pellets, six different end-user PVC-based products including three widely used PVC water pipes were further examined. Among them, extracts of PVC pipe exerted the strongest PPARγ activity and cell viability suppression. Organotins were identified as the primary drivers to these in vitro toxicities induced by the PVC pipe extracts. This study clearly delineates specific chemical additives responsible for hCES1 inhibition, PPARγ activity, and cell viability suppression associated with plastic.

Quantitative Chemical Proteomics Reveals Interspecies Variations on Binding Schemes of L-FABP with Perfluorooctanesulfonate.

Evaluating interspecies toxicity variation is a long-standing challenge for chemical hazard assessment. This study developed a quantitative interspecies thermal shift assay (QITSA) for in situ, quantitative, and modest-throughput investigation of chemical-protein interactions in cell and tissue samples across species. By using liver fatty acid binding protein (L-FABP) as a case study, the QITSA method was benchmarked with six per- and polyfluoroalkyl substances, and thermal shifts (ΔTm) were inversely related to their dissociation constants (R2 = 0.98). The QITSA can also distinguish binding modes of chemicals exemplified by palmitic acid. The QITSA was applied to determine the interactions between perfluorooctanesulfonate (PFOS) and L-FABP in liver cells or tissues from humans, mice, rats, and zebrafish. The largest thermal stability enhancement by PFOS was observed for human L-FABP followed by the mouse, rat, and zebrafish. While endogenous ligands were revealed to partially contribute to the large interspecies variation, recombinant proteins were employed to confirm the high binding affinity of PFOS to human L-FABP, compared to the rat and mouse. This study implemented an experimental strategy to characterize chemical-protein interactions across species, and future application of QITSA to other chemical contaminants is of great interest.

Potential Link between Equol Pollution and Field-Observed Intersex in Wild So-iuy Mullets (Mugil soiuy).

Gonadal intersex has been observed in wild fishes and is attributed to endocrine-disrupting chemicals but the specific causes remain controversial. Here, a forensic analysis utilizing field and laboratory studies was conducted to explore the causal agent(s). In a 2008-2009 survey of Liaodong Bay, China, 20.7-33.3% incidences of gonadal intersex were observed in male so-iuy mullets (Mugil soiuy), a wild sentinel fish species. Steroidal estrogen (estrone, 17ß-estradiol, estriol, and ethinylestradiol) and phytoestrogen (equol) were detected in seawater where the fishes were collected with median concentrations of 0.42 ng/L (0.02-1.42 ng/L) E2 equivalent (EEQ-E2) and 22.81 ng/L (0.10-155.99 ng/L) equol. A probabilistic model was used to evaluate the ecological risk of these estrogenic chemicals based on their distribution in the field and dose-response relationship from the laboratory surrogate Japanese medaka (Oryzias latipes) fish. The probability of the incidences of gonadal intersex due to equol exposure was estimated to be 13.5 ± 12.1%, which is considerably higher than that for EEQ-E2, (7.2 ± 68.8) × 10-4. The agonistic activity of equol to the estrogen receptor α of so-iuy mullets was 3.5-fold higher than that to the estrogen receptor α of Japanese medaka, indicating that equol shows a stronger potential for inducing intersex in so-iuy mullets than in medaka. These results demonstrate that equol, rather than steroid estrogens, is a more likely causal agent for the field-observed intersex in male wild so-iuy mullets.

Toxicokinetics of Brominated Azo Dyes in the Early Life Stages of Zebrafish (Danio rerio) Is Prone to Aromatic Substituent Changes.

Brominated azo dyes (BADs) have been identified as predominant indoor brominated pollutants in daycare dust; thus, their potential health risk to children is of concern. However, the toxicities of BADs remain elusive. In this study, the toxicokinetics of two predominant BADs, Disperse Blue 373 (DB373) and Disperse Violet 93 (DV93), and their suspect metabolite 2-bromo-4,6-dinitroaniline (BDNA) was investigated in embryos of zebrafish (Danio rerio). The bioconcentration factor of DV93 at 120 hpf is 6.2-fold lower than that of DB373. The nontarget analysis revealed distinct metabolism routes between DB373 and DV93 by reducing nitro groups to nitroso (DB373) or amine (DV93), despite their similar structures. NAD(P)H quinone oxidoreductase 1 (NQO1) and pyruvate dehydrogenase were predicted as the enzymes responsible for the reduction of DB373 and DV93 by correlating time courses of the metabolites and enzyme development. Further in vitro recombinant enzyme and in vivo inhibition results validated NQO1 as the enzyme specifically reducing DB373, but not DV93. Global proteome profiling revealed that the expression levels of proteins from the "apoptosis-induced DNA fragmentation" pathway were significantly upregulated by all three BADs, supporting the bioactivation of BADs to mutagenic aromatic amines. This study discovered the bioactivation of BADs via distinct eukaryotic enzymes, implying their potential health risks.

Nontarget Screening of Per- and Polyfluoroalkyl Substances Binding to Human Liver Fatty Acid Binding Protein.

More than 1000 per- and polyfluoroalkyl substances (PFASs) have been discovered by nontarget analysis (NTA), but their prioritization for health concerns is challenging. We developed a method by incorporating size-exclusion column co-elution (SECC) and NTA, to screen PFASs binding to human liver fatty acid binding protein (hL-FABP). Of 74 PFASs assessed, 20 were identified as hL-FABP ligands in which eight of them have high binding affinities. Increased PFAS binding affinities correlate with stronger responses in electrospray ionization (ESI-) and longer retention times on a C18 column. This is well explained by a mechanistic model, which revealed that both polar and hydrophobic interactions are crucial for binding affinities. Encouraged by this, we then developed an SECC method to identify hL-FABP ligands, and all eight high-affinity ligands were selectively captured from 74 PFASs. The method was further applied to an aqueous film-forming foam (AFFF) product in which 31 new hL-FABP ligands were identified. Suspect and nontargeted screening revealed these ligands as analogues of perfluorosulfonic acids and homologues of alkyl ether sulfates (C8- and C10/EOn, C8H17(C2H4O)nSO4-, and C10H21(C2H4O)nSO4-). The SECC method was then applied to AFFF-contaminated surface waters. In addition to perfluorooctanesulfonic acid and perfluorohexanesulfonic acid, eight other AFFF chemicals were discovered as novel ligands, including four C14- and C15/EOn. This study implemented a high-throughput method to prioritize PFASs and revealed the existence of many previously unknown hL-FABP ligands.

Identification of Chemicals that Cause Oxidative Stress in Oil Sands Process-Affected Water.

Oil sands process-affected water (OSPW) has been reported to cause oxidative stress in organisms, yet the causative agents remain unknown. In this study, a high-throughput in vitro Nrf2 reporter system was used, to determine chemicals in OSPW that cause oxidative stress. Five fractions, with increasing polarity, of the dissolved organic phase of OSPW were generated by use of solid phase extraction cartridges. The greatest response of Nrf2 was elicited by F2 (2.7 ± 0.1-fold), consistent with greater hydroperoxidation of lipids in embryos of Japanese medaka (Oryzias latipes) exposed to F2. Classic naphthenic acids were mainly eluted in F1, and should not be causative chemicals. When F2 was fractionated into 60 subfractions by use of HPLC, significant activation of Nrf2 was observed in three grouped fractions: F2.8 (1.30 ± 0.01-fold), F2.16 (1.34 ± 0.05-fold), and F2.25 (1.28 ± 0.15-fold). 54 compounds were predicted to be potential chemicals causing Nrf2 response, predominated by SO3+ and O3+ species. By use of high-resolution MS2 spectra, these SO3+ and O3+ species were identified as hydroxylated aldehydes. This study demonstrated that polyoxygenated chemicals, rather than classic NAs, were the major chemicals responsible for oxidative stress in the aqueous phase of OSPW.

p,p'-DDE Induces Gonadal Intersex in Japanese Medaka (Oryzias latipes) at Environmentally Relevant Concentrations: Comparison with o,p'-DDT.

Previous studies have reported high body burdens of dichlorodiphenyltrichloroethane (DDT) and its metabolites in wild fishes worldwide. This study evaluated the adverse effects of 1,1-dichloro-2,2-bis (p-chlorophenyl)-ethylene (p,p'-DDE) and o,p'-DDT on gonadal development and reproduction by exposing transgenic Japanese medaka (Oryzias latipes) from hatch for 100 days. While both p,p'-DDE and o,p'-DDT induced intersex in male medaka, the lowest observable effective concentration (LOEC) of o,p'-DDT was 57.7 ng/g ww, about 5-fold lower than that (272 ng/g ww) of p,p'-DDE. Since LOECs of both chemicals were comparable to the body concentrations in wild fish, DDT contamination would likely contribute to the occurrence of intersex observed in wild fish. Exposure to o,p'-DDT resulted in much higher expression of vitellogenin in liver of males than p,p'-DDE, accordant with the higher potency of o,p'-DDT than p,p'-DDE to induce intersex. This phenomenon could be partly explained by the significantly elevated levels of 17ß-estradiol in plasma of males exposed to o,p'-DDT, in addition to its estrogenic activity via the estrogen receptor. Significantly lower fertilization (p = 0.006) and hatchability (p = 0.019) were observed in the 13 intersex males. This study for the first time demonstrated the induction of intersex and reproductive effects of p,p'-DDE and o,p'-DDT at environmentally relevant concentrations.

Peroxisome Proliferator-Activated Receptor γ is a Sensitive Target for Oil Sands Process-Affected Water: Effects on Adipogenesis and Identification of Ligands.

Identification of toxic components of complex mixtures is a challenge. Here, oil sands process-affected water (OSPW) was used as a case study to identify those toxic components with a known protein target. Organic chemicals in OSPW exhibited dose-dependent activation of peroxisome proliferator-activated receptor γ (PPARγ) at concentrations less than those currently in the environment (0.025× equivalent of full-strength OSPW), by use of a luciferase reporter gene assay. Activation of PPARγ-mediated adipogenesis by OSPW was confirmed in 3T3L1 preadipocytes, as evidenced by accumulation of lipids and up-regulation of AP2, LPL, and PPARγ gene expression after exposure to polar fractions of OSPW. Unexpectedly, the nonpolar fractions of OSPW inhibited differentiation of preadipocytes via activation of the Wnt signaling pathway. Organic chemicals in OSPW that were ligands of PPARγ were identified by use of a pull-down system combined with untargeted chemical analysis (PUCA), with a recombinant PPARγ protein. Thirty ligands of PPARγ were identified by use of the PUCA assay. High resolution MS(1) and MS(2) spectra were combined to predict the formulas or structures of a subset of ligands, and polyoxygenated or heteroatomic chemicals, especially hydroxylated carboxylic/sulfonic acids, were the major ligands of PPARγ.

Combined Transcriptomic and Proteomic Approach to Identify Toxicity Pathways in Early Life Stages of Japanese Medaka (Oryzias latipes) Exposed to 1,2,5,6-Tetrabromocyclooctane (TBCO).

Currently, the novel brominated flame retardant 1,2,5,6-tetrabromocyclooctane (TBCO) is considered a potential replacement for hexabromocyclododecane (HBCD). Therefore, use of TBCO could increase in the near future. To assess potential toxicological risks to aquatic organisms, embryos of Japanese medaka (Oryzias latipes) were exposed to 10, 100, or 1000 µg/L TBCO from 2 h postfertilization until 1 day post-hatch. TBCO accumulated in embryos in the order of 0.43-1.3 × 10(4)-fold, and the rate constant of accumulation was 1.7-1.8 per day. The number of days to hatch and the hatching success of embryos exposed to the medium and the greatest concentrations of TBCO were impaired. Responses of the transcriptome (RNA-seq) and proteome were characterized in embryos exposed to 100 µg/L TBCO because this was the least concentration of TBCO that caused an effect on hatching. Consistent with effects on hatching, proteins whose abundances were reduced by exposure to TBCO were enriched in embryo development and hatching pathways. Also, on the basis of the responses of transcriptome and proteome, it was predicted that TBCO might impair vision and contraction of cardiac muscle, respectively, and these effects were confirmed by targeted bioassays. This study provided a comprehensive understanding of effects of TBCO on medaka at early life stages and illustrated the power of "omics" to explain and predict phenotypic responses to chemicals.

High Conservation in Transcriptomic and Proteomic Response of White Sturgeon to Equipotent Concentrations of 2,3,7,8-TCDD, PCB 77, and Benzo[a]pyrene.

Adverse effects associated with exposure to dioxin-like compounds (DLCs) are mediated primarily through activation of the aryl hydrocarbon receptor (AHR). However, little is known about the cascades of events that link activation of the AHR to apical adverse effects. Therefore, this study used high-throughput, next-generation molecular tools to investigate similarities and differences in whole transcriptome and whole proteome responses to equipotent concentrations of three agonists of the AHR, 2,3,7,8-TCDD, PCB 77, and benzo[a]pyrene, in livers of a nonmodel fish, the white sturgeon (Acipenser transmontanus). A total of 926 and 658 unique transcripts were up- and down-regulated, respectively, by one or more of the three chemicals. Of the transcripts shared by responses to all three chemicals, 85% of up-regulated transcripts and 75% of down-regulated transcripts had the same magnitude of response. A total of 290 and 110 unique proteins were up- and down-regulated, respectively, by one or more of the three chemicals. Of the proteins shared by responses to all three chemicals, 70% of up-regulated proteins and 48% of down-regulated proteins had the same magnitude of response. Among treatments there was 68% similarity between the global transcriptome and global proteome. Pathway analysis revealed that perturbed physiological processes were indistinguishable between equipotent concentrations of the three chemicals. The results of this study contribute toward more completely describing adverse outcome pathways associated with activation of the AHR.

Mutagenic Azo Dyes, Rather Than Flame Retardants, Are the Predominant Brominated Compounds in House Dust.

Characterization of toxicological profiles by use of traditional targeted strategies might underestimate the risk of environmental mixtures. Unbiased identification of prioritized compounds provides a promising strategy for meeting regulatory needs. In this study, untargeted screening of brominated compounds in house dust was conducted using a data-independent precursor isolation and characteristic fragment (DIPIC-Frag) approach, which used data-independent acquisition (DIA) and a chemometric strategy to detect peaks and align precursor ions. A total of 1008 brominated compound peaks were identified in 23 house dust samples. Precursor ions and formulas were identified for 738 (73%) of the brominated compounds. A correlation matrix was used to cluster brominated compounds; three large groups were found for the 140 high-abundance brominated compounds, and only 24 (17%) of these compounds were previously known flame retardants. The predominant class of unknown brominated compounds was predicted to consist of nitrogen-containing compounds. Following further validation by authentic standards, these compounds (56%) were determined to be novel brominated azo dyes. The mutagenicity of one major component was investigated, and mutagenicity was observed at environmentally relevant concentrations. Results of this study demonstrated the existence of numerous unknown brominated compounds in house dust, with mutagenic azo dyes unexpectedly being identified as the predominant compounds.

Linking Oxidative Stress and Magnitude of Compensatory Responses with Life-Stage Specific Differences in Sensitivity of White Sturgeon (Acipenser transmontanus) to Copper or Cadmium.

Sensitivity of white sturgeon (Acipenser transmontanus) to copper (Cu) or cadmium (Cd) has been shown to significantly differ as a function of life-stage. This study investigated oxidative stress, metal homeostasis, and associated compensatory responses as potential mechanisms of this sensitivity pattern in three early life-stages. Sturgeon were most sensitive to Cu at 15 days post hatch (dph), which was accompanied by a significant increase in lipid peroxidation (LPO). Genes involved with amelioration of oxidative stress were significantly less inducible at this stage than in older, less sensitive fry. At 48 dph, acute lethality of sturgeon exposed to Cd was greatest and body LPO was significantly induced by 3.5-fold at 5 µg Cd/L. Moreover, there was a small but significant increase in antioxidative responses. At 139 dph, sturgeon were most tolerant to Cu and Cd and accumulation of these metals was least. Also, expression of metallothionein (MT) and apoptotic genes were greatest while expression of metal transporters was reduced and concentration of LPO was not different from controls. Our results suggest that life-stage specific sensitivity of white sturgeon to metals is complex, encompassing differences in the ability to mount compensatory responses important for metal homeostasis and combating oxidative stress and concomitant damages.

Untargeted Screening and Distribution of Organo-Bromine Compounds in Sediments of Lake Michigan.

Previously unreported natural and synthetic organo-bromine compounds (NSOBCs) have been found to contribute more than 99% of total organic bromine (TOB) in environmental matrices. We recently developed a novel untargeted method (data-independent precursor isolation and characteristic fragment, DIPIC-Frag) and identified ∼2000 NSOBCs in two sediments from Lake Michigan. In this study, this method was used to investigate the distributions of these NSOBCs in 23 surficial samples and 24 segments of a sediment core from Lake Michigan. NSOBCs were detected in all 23 surficial samples and exhibited 10- to 100-fold variations in peak abundance among locations. The pattern of distributions of NSOBCs was correlated with depth of the water column (r(2) = 0.61, p < 0.001). Hierarchical cluster analysis showed that sediments in close proximity exhibited similar profiles of NSOBCs. Distributions of NSOBCs in 24 segments of a sediment core dated from 1766 to 2008 were investigated, and samples from similar depths exhibited similar profiles of NSOBCs. NSOBCs were grouped into four clusters (soft-cluster analysis) with different temporal trends of abundances. 515 and 768 of the NSOBCs were grouped into cluster 1 and cluster 3 with increasing temporal trends, especially since 1950, indicating that abundances of these compounds might have been affected by human activities.