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Chemical modifications of proteins, DNA, and RNA moieties play critical roles in regulating gene expression. Emerging evidence suggests the RNA modifications (epitranscriptomics) have substantive roles in basic biological processes. One of the most common modifications in mRNA and noncoding RNAs is N6-methyladenosine (m6A). In a subset of mRNAs, m6A sites are preferentially enriched near stop codons, in 3' UTRs, and within exons, suggesting an important role in the regulation of mRNA processing and function including alternative splicing and gene expression. Very little is known about the effect of environmental chemical exposure on m6A modifications. As many of the commonly occurring environmental contaminants alter gene expression profiles and have detrimental effects on physiological processes, it is important to understand the effects of exposure on this important layer of gene regulation. Hence, the objective of this study was to characterize the acute effects of developmental exposure to PCB126, an environmentally relevant dioxin-like PCB, on m6A methylation patterns. We exposed zebrafish embryos to PCB126 for 6 h starting from 72 h post fertilization and profiled m6A RNA using methylated RNA immunoprecipitation followed by sequencing (MeRIP-seq). Our analysis revealed 117 and 217 m6A peaks in the DMSO and PCB126 samples (false discovery rate 5%), respectively. The majority of the peaks were preferentially located around the 3' UTR and stop codons. Statistical analysis revealed 15 m6A marked transcripts to be differentially methylated by PCB126 exposure. These include transcripts that are known to be activated by AHR agonists (eg, ahrra, tiparp, nfe2l2b) as well as others that are important for normal development (vgf, cebpd, sned1). These results suggest that environmental chemicals such as dioxin-like PCBs could affect developmental gene expression patterns by altering m6A levels. Further studies are necessary to understand the functional consequences of exposure-associated alterations in m6A levels., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Polychlorinated biphenyls (PCBs) are highly persistent and ubiquitously distributed environmental pollutants. Based on their chemical structure, PCBs are classified into non-ortho-substituted and ortho-substituted congeners. Non-ortho-substituted PCBs are structurally similar to dioxin and their toxic effects and mode of action are well-established. In contrast, very little is known about the effects of ortho-substituted PCBs, particularly, during early development. The objective of this study is to investigate the effects of exposure to an environmentally prominent ortho-substituted PCB (2,2',4,4',5,5'-hexachlorobiphenyl; PCB153) on zebrafish embryos. We exposed zebrafish embryos to 3 different concentrations of PCB153 starting from 4 to 120 hours post-fertilization (hpf). We quantified gross morphological changes, behavioral phenotypes, gene expression changes, and circadian behavior in the larvae. There were no developmental defects during the exposure period, but starting at 7 dpf, we observed spinal deformity in the 10 μM PCB153 treated group. A total of 633, 2227, and 3378 differentially expressed genes were observed in 0.1 μM (0.036 μg/ml), 1 μM (0.36 μg/ml), and 10 μM (3.6 μg/ml) PCB153-treated embryos, respectively. Of these, 301 genes were common to all treatment groups. KEGG pathway analysis revealed enrichment of genes related to circadian rhythm, FoxO signaling, and insulin resistance pathways. Behavioral analysis revealed that PCB153 exposure significantly alters circadian behavior. Disruption of circadian rhythms has been associated with the development of metabolic and neurological diseases. Thus, understanding the mechanisms of action of environmental chemicals in disrupting metabolism and other physiological processes is essential., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Interactions between regulatory pathways allow organisms to adapt to their environment and respond to stress. One interaction that has been recently identified occurs between the aryl hydrocarbon receptor (AHR) and the nuclear factor erythroid-2 related factor (NRF) family. Each transcription factor regulates numerous downstream genes involved in the cellular response to toxicants and oxidative stress; they are also implicated in normal developmental pathways. The zebrafish model was used to explore the role of AHR regulation of nrf genes during development and in response to toxicant exposure. To determine if AHR1b is responsible for transcriptional regulation of 6 nrf genes during development, a loss-of-function experiment using morpholino-modified oligonucleotides was conducted followed by a chromatin immunoprecipitation study at the beginning of the pharyngula period (24 h postfertilization). The expression of nrf1a was AHR1b dependent and its expression was directly regulated through specific XREs in its cis-promoter. However, nrf1a expression was not altered by exposure to 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), a toxicant and prototypic AHR agonist. The expression of nrf1b, nrf2a, and nfe2 was induced by TCDD, and AHR1b directly regulated their expression by binding to cis-XRE promoter elements. Last, nrf2b and nrf3 were neither induced by TCDD nor regulated by AHR1b. These results show that AHR1b transcriptionally regulates nrf genes under toxicant modulation via binding to specific XREs. These data provide a better understanding of how combinatorial molecular signaling potentially protects embryos from embryotoxic events following toxicant exposure.

Early life exposure to environmental chemicals can have long-term consequences that are not always apparent until later in life. We recently demonstrated that developmental exposure of zebrafish to low, nonembryotoxic levels of 3,3',4,4',5-pentachlorobiphenyl (PCB126) did not affect larval behavior, but caused changes in adult behavior. The objective of this study was to investigate the underlying molecular basis for adult behavioral phenotypes resulting from early life exposure to PCB126. We exposed zebrafish embryos to PCB126 during early development and measured transcriptional profiles in whole embryos, larvae and adult male brains using RNA-sequencing. Early life exposure to 0.3 nM PCB126 induced cyp1a transcript levels in 2-dpf embryos, but not in 5-dpf larvae, suggesting transient activation of aryl hydrocarbon receptor with this treatment. No significant induction of cyp1a was observed in the brains of adults exposed as embryos to PCB126. However, a total of 2209 and 1628 genes were differentially expressed in 0.3 and 1.2 nM PCB126-exposed groups, respectively. KEGG pathway analyses of upregulated genes in the brain suggest enrichment of calcium signaling, MAPK and notch signaling, and lysine degradation pathways. Calcium is an important signaling molecule in the brain and altered calcium homeostasis could affect neurobehavior. The downregulated genes in the brain were enriched with oxidative phosphorylation and various metabolic pathways, suggesting that the metabolic capacity of the brain is impaired. Overall, our results suggest that PCB exposure during sensitive periods of early development alters normal development of the brain by reprogramming gene expression patterns, which may result in alterations in adult behavior., (© The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

The sensitivity of avian species to the toxic effects of dioxin-like compounds (DLCs) varies up to 1000-fold among species, and this variability has been associated with interspecies differences in aryl hydrocarbon receptor 1 ligand-binding domain (AHR1 LBD) sequence. We previously showed that LD(50) values, based on in ovo exposures to DLCs, were significantly correlated with in vitro EC(50) values obtained with a luciferase reporter gene (LRG) assay that measures AHR1-mediated induction of cytochrome P4501A in COS-7 cells transfected with avian AHR1 constructs. Those findings suggest that the AHR1 LBD sequence and the LRG assay can be used to predict avian species sensitivity to DLCs. In the present study, the AHR1 LBD sequences of 86 avian species were studied, and differences at amino acid sites 256, 257, 297, 324, 337, and 380 were identified. Site-directed mutagenesis, the LRG assay, and homology modeling highlighted the importance of each amino acid site in AHR1 sensitivity to 2,3,7,8-tetrachlorodibenzo-p-dioxin and other DLCs. The results of the study revealed that (1) only amino acids at sites 324 and 380 affect the sensitivity of AHR1 expression constructs of the 86 avian species to DLCs and (2) in vitro luciferase activity of AHR1 constructs containing only the LBD of the species of interest is significantly correlated (r (2) = 0.93, p < 0.0001) with in ovo toxicity data for those species. These results indicate promise for the use of AHR1 LBD amino acid sequences independently, or combined with the LRG assay, to predict avian species sensitivity to DLCs.

The aryl hydrocarbon receptor (AHR) repressor (AHRR), an AHR-related basic helix-loop-helix/Per-AHR nuclear translocator-Sim protein, is regulated by an AHR-dependent mechanism and acts as a transcriptional repressor of AHR function. Resulting from a teleost-specific genome duplication, zebrafish have two AHRR genes (AHRRa and AHRRb), but their functions in vivo are not well understood. We used antisense morpholino oligonucleotides (MOs) in zebrafish embryos and a zebrafish liver cell line (ZF-L) to characterize the interaction of AHRRs and AHRs in normal embryonic development, AHR signaling, and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity. Zebrafish embryos exposed to TCDD (2 and 8nM) during early development showed strong induction of CYP1A, AHRRa, and AHRRb at 48 and 72 hours post-fertilization (hpf). An MO targeting AHR2 inhibited TCDD-induced expression of CYP1A, AHRRa, and AHRRb by 84-95% in 48 hpf embryos, demonstrating a primary role for AHR2 in mediating AHRR induction. Dual MO knockdown of both AHRRs in ZF-L cells enhanced TCDD induction of CYP1A, but not other CYP1 genes. In embryos, dual knockdown of AHRRs, or knockdown of AHRRb alone, enhanced the induction of CYP1A, CYP1B1, and CYP1C1 by TCDD and decreased the constitutive expression of Sox9b. In contrast, knockdown of AHRRa did not affect Sox9b expression or CYP1 inducibility. Embryos microinjected with each of two different MOs targeting AHRRa and exposed to dimethyl sulfoxide (DMSO) displayed developmental phenotypes resembling those typical of TCDD-exposed embryos (pericardial edema and lower jaw malformations). In contrast, no developmental phenotypes were observed in DMSO-exposed AHRRb morphants. These data demonstrate distinct roles of AHRRa and AHRRb in regulating AHR signaling in vivo and suggest that they have undergone subfunction partitioning since the teleost-specific genome duplication.

Dioxins including 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induce various toxic effects through the aryl hydrocarbon receptor (AhR) signaling pathway. Here, we investigated the structural and functional characteristics and molecular evolution of multiple AhRs in black-footed albatross (Phoebastria nigripes) and common cormorant (Phalacrocorax carbo). We report the complementary DNA sequences of two distinct AhRs, designated AhR1 and AhR2, from these species as well as the identification of an AhR2-like gene sequence from the chicken genome database. Phylogenetic analysis reveals that avian AhR1 and AhR2 are orthologous to mammalian AhR1 and fish AhR2, respectively, supporting the hypothesis that an ancestral AhR gene underwent a tandem duplication prior to the divergence of fish and tetrapod lineages. In vitro-expressed AhR1 and AhR2 isoforms from both albatross and cormorant exhibited specific binding to [3H]TCDD, as assessed by velocity sedimentation. An in vitro reporter gene transactivation assay revealed that both AhR1 and AhR2 are transcriptionally active, but AhR2 appears to have reduced transcriptional efficacy. Hepatic messenger RNA expression level of cormorant AhR1 was greater than that of AhR2. Together, these results suggest that AhR1 is the dominant form of avian AhRs, in contrast to fish, in which AhR2 is the major form. Comparative analysis of AhR diversity and gene synteny among chicken, zebrafish, and human suggests that additional, independent AhR duplications have occurred in the fish and tetrapod lineages following the initial tandem duplication on the ancestral chromosome. The identification and characterization of avian AhR1 and AhR2 provide new insight into the evolution of AhR structure and function in vertebrates.

Several studies have reported a low inducibility of hepatic cytochrome P4501A (CYP1A) activity in European flounder (Platichthys flesus) following exposure to mixtures of polychlorinated biphenyls (PCBs). Here we report on mechanistic studies toward understanding this low CYP1A inducibility of flounder, involving molecular characterization of the Ah receptor (AhR) pathway as well as inhibition of the CYP1A catalytic activity by PCB congeners. Hepatic cytosolic AhR levels in flounder were determined using hydroxylapatite, protamine sulfate adsorption analysis, or velocity sedimentation on sucrose gradients. AhR levels in flounder (approximately 2-7 fmol/mg protein) were much lower than observed generally in rodents (approximately 50-300 fmol/mg protein). Molecular characterization of the flounder AhR was provided by first-strand cDNA synthesis and amplification of flounder hepatic poly(A)+ RNA using RT-PCR. A 690-bp product was found, similar in size to a Fundulus AhR cDNA. The specificity of the 690-bp band was established by Southern blotting and hybridization with a degenerate AhR oligonucleotide. The deduced amino acid sequence of the flounder AhR fragment was 59-60% identical to mammalian AhR sequences. Although the AhR is present in flounder cytosol, we were unable to demonstrate detectable amounts of inducible TCDD-AhR-DRE complex in gel-retardation assays. High induction levels of CYP1A protein and associated EROD activity have been previously found in flounder following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In contrast, the induction of CYP1A catalytic activity by PCB mixtures remains unexpectedly low. Therefore, we further characterized the inhibitory potential of PCB congeners on CYP1A activity in flounder and compared this with inhibitory effects of PCB congeners on rat CYP1A activity. Analysis in vitro demonstrated that 3,3',4,4'-tetraCB, 3,3',4,4',5-pentaCB, 2,2',4,4',5,5'-hexaCB, 3,3',4,4',5,5'-hexaCB, and the commercial PCB mixture Clophen A50 are potent competitive inhibitors of hepatic microsomal CYP1A catalytic activity in flounder and rat. The K(m) for ethoxyresorufin (0.095 microM) in flounder is strikingly close to Ki's found for the tested PCBs. This emphasizes the possible involvement of PCB congeners in inhibition of EROD activity in PHAH exposed fish. Finally, our data indicate that flounder CYP1A is more efficient in metabolizing ethoxyresorufin than that of rat CYP1A.