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Published in final edited form as: Aquat Toxicol. 2022 Jun 9;249:106219. doi: 10.1016/j.aquatox.2022.106219

Nrf2a dependent and independent effects of early life exposure to 3,3’-dichlorobiphenyl (PCB-11) in zebrafish (Danio rerio)

Monika A Roy 1,2, Charlotte K Gridley 1, Sida Li 3, Yeonhwa Park 3, Alicia R Timme-Laragy 1
PMCID: PMC9701526  NIHMSID: NIHMS1848551  PMID: 35700651

Abstract

The environmental pollutant 3,3’-dichlorobiphenyl (PCB-11) is a lower-chlorinated polychlorinated biphenyl (PCB) congener present in air and water samples. Both PCB-11 and its metabolite, 4-PCB-11-Sulfate, are detected in humans, including in pregnant women. Previous research in zebrafish (Danio reno) has shown that 0.2 μM exposures to 4-PCB-11-Sulfate starting at 1 day post fertilization (dpf) increase hepatic neutral lipid accumulation in larvae at 15 dpf. Here, we explored whether nuclear factor erythroid 2-related factor 2 (Nrf2), known as the master-regulator of the adaptive response to oxidative stress, contributes to metabolic impacts of 4-PCB-11-Sulfate. For this work, embryos were collected from homozygous wildtype or Nrf2a mutant adult zebrafish that also express GFP in pancreatic β-cells, rendering Tg(ins:GFP;nrf2afh318+/+) and Tg(ins:GFP;nrf2afh318−/−) lines. Exposures were conducted from 1-15 dpf to either 0.05% DMSO or DMSO-matched 0.2 μM 4-PCB-11-Sulfate, and at 15 dpf subsets of larvae were imaged for overall morphology, primary pancreatic islet area, and collected for fatty acid profiling and RNAseq. At 15 dpf, independent of genotype, fish exposed to 4-PCB-11-Sulfate survived significantly more at 80-85% compared to 65-73% survival for unexposed fish, and had primary pancreatic islets 8% larger compared to unexposed fish. Fish growth at 15 dpf was dependent on genotype, with Nrf2a mutant fish a significant 3-5% shorter than wildtype fish, and an interaction effect was observed where Nrf2a mutant fish exposed to 4-PCB-11-Sulfate experienced a significant 29% decrease in the omega-3 fatty acid DHA compared to unexposed mutant fish. RNAseq revealed 308 differentially expressed genes, most of which were dependent on genotype. These findings suggest that Nrf2a plays an important role in growth as well as for DHA production in the presence of 4-PCB-11-Sulfate. Further research would be beneficial to understand the importance of Nrf2a throughout the lifecourse, especially in the context of toxicant exposures.

Keywords: PCB-11, zebrafish, Nrf2, fatty acid profiling, pancreas, RNAseq

1. Introduction

The environmental pollutant 3,3’-dichlorobiphenyl (PCB-11) is a semi-volatile polychlorinated biphenyl (PCB) widely detected in indoor and outdoor air samples (Herkert et al. 2018; Shanahan et al. 2015) in the U.S. and worldwide (Guo et al. 2014; Hao et al. 2021). PCB-11 is most notably generated as an inadvertent byproduct in industrial azo-type yellow pigment manufacturing through the use of its parent compound 3,3’-dichlorobenzidine, where PCB-11 is released into the environment during manufacturing or later through the breakdown of products it is found in (Hu and Hornbuckle 2010; Vorkamp 2016). Inhalation is a major route of exposure for humans, and both PCB-11 and one of its primary metabolites, 4-PCB-11-Sulfate, have been detected in human serum, including in pregnant women (Grimm et al. 2017; Koh et al. 2015; Sethi et al. 2017). Higher-chlorinated non-ortho-substituted (coplanar) PCBs are well-established as causing dioxin-like toxicity to humans, however, very little in vivo toxicity data exists examining lower-chlorinated PCBs like PCB-11, which is also non-ortho-substituted and may assume a degree of planarity.

Coplanar PCBs are well-characterized as agonists of the Aryl hydrocarbon receptor (Ahr) pathway, that when activated can either catalyze toxicity or neutralize the agonist. During Ahr pathway activation, many genes responsible for metabolizing the agonist are upregulated, such as cytochrome p450 (Cyp) 1a, or cyp1a, which is then translated to its enzyme counterpart Cyp1a. Zebrafish cyp1a is the most highly inducible member of the Cyp1 family and is similar to human Cyp1a1 and Cyp1a2 induction via the Ahr pathway (Jonsson et al. 2007; Nebert et al. 2004; Saad et al. 2016). Importantly, when Cyp1a enzyme activity is uncoupled, this can generate reactive oxygen species and oxidative stress (Schlezinger et al. 2006), contributing to crosstalk between the Ahr and the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway (Wakabayashi et al. 2010; Yeager et al. 2009). Previous work demonstrated that both PCB-11 and 4-PCB-11-Sulfate inhibit Cyp1a enzyme activity (Roy et al. 2020; Roy et al. 2019), so a particular interest for this study was whether 4-PCB-11-Sulfate interacts with the Nrf2 pathway.

Nrf2 is a master-regulator of the adaptive response to oxidative stress, employing glutathione (GSH) in its reduced and oxidized disulfide (GSSG) form as a redox buffering system to manage both endogenous fluctuations in reactive oxygen species (ROS) as well as exogenous environmental chemicals that increase ROS (Hansen and Harris 2013; Sies et al. 2017). Increased cytosolic ROS levels stimulate Nrf2 nuclear translocation to bind with the antioxidant response element (ARE), upregulating a battery of target genes to support a return to a homeostatic redox range (Lu 2013; Nguyen et al. 2003). Nrf2 also plays an important role in embryonic development through the tight control of redox states at specific stages of cell proliferation and differentiation (Timme-Laragy et al. 2013; Timme-Laragy et al. 2018). Zebrafish are a useful model for probing Nrf2, and though genome duplication in zebrafish has given rise to nrf2a and nrf2b co-orthologs, nrf2a is the canonical activator of the ARE in response to ROS, while nrf2b plays a negative regulatory role of several key genes during development (Timme-Laragy et al. 2012). Zebrafish with a point mutation in the DNA-binding domain of Nrf2a (Mukaigasa et al. 2012) and that have transgenic labels in specific cells are available for studying the role of Nrf2a in specific organs in response to environmental chemicals (Jacobs et al. 2018; Rastogi et al. 2021).

In addition to the inhibition of hepatic Cyp1a enzyme activity, our previous work in zebrafish has shown that chronic exposures to 0.2 μM 4-PCB-11-Sulfate from 1-15 dpf increase hepatic neutral lipid accumulation (Roy et al. 2020). As has been shown in rodents, PCB-11 undergoes metabolism and distributes to many tissues (Grimm et al. 2015; Hu et al. 2014), but primarily to adipose tissue (Zhang et al. 2021), not only a major place of lipid storage and metabolism, but also often a site of inflammation and reactive oxygen species production (Han 2016). Non-dioxin-like PCBs in general have been shown to be metabolism-disrupting compounds, associated with dyslipidemia, steatosis, and disrupted leptin and pancreatic insulin production, among other effects (Wahlang et al. 2019). Our research group has shown that pancreatic β-cells are exquisitely sensitive to developmental redox disruptions (Rastogi et al, 2021), prompting the concern that PCB-11 may disrupt organogenesis of the islet in addition to other potential metabolic effects. In this study, we investigated the dependent and independent effects of Nrf2a on survival, pancreas development, fatty acid profiles, and gene expression after exposures to 0.2 μM 4-PCB-11-Sulfate between 1-15 dpf. Our main objectives were to explore the role of Nrf2a on fish development, and to investigate the role of Nrf2a on metabolic-related endpoints in the presence of a prevalent environmental chemical metabolite like 4-PCB-11-Sulfate.

2. Materials and Methods

2.1. Animal Care

Zebrafish (Danio rerio) with a loss-of-function nrf2afh318−/− mutation (Mukaigasa et al. 2012) were generated through the TILLING mutagenesis project (R01 HD076585) and obtained as homozygous embryos from Dr. Mark Hahn at the Woods Hole Oceanographic Institution. Genotypes were confirmed by PCR as described elsewhere (Rousseau et al. 2015). Previously in the Timme-Laragy lab, wildtype and mutant nrf2afh318−/− zebrafish were crossed with Tg(ins:GFP) transgenic zebrafish with an AB strain background expressing GFP in pancreatic β-cells to create the Tg(ins:GFP;nrf2afh318+/+) and Tg(ins:GFP;nrf2afh318−/−) zebrafish lines. These two zebrafish lines were used for experiments described in this manuscript, and for simplicity are referred to as “Nrf2a wildtype” for the homozygous wildtype Tg(ins:GFP;nrf2afh318+/+) line and “Nrf2a mutant” for the homozygous mutant Tg(ins:GFP;nrf2afh318−/−) zebrafish line in the figures.

Adult zebrafish were housed on a 14:10 h light:dark cycle in a recirculating Aquaneering system (San Diego, CA) maintained at 28.5°C and fed twice daily with GEMMA Micro 300 (Skretting, Westbrook, ME). Embryos were collected from tanks with a 2:1 female:male ratio within 3 hours of breeding, and then were rinsed and stored at low density in 0.3x Danieau’s media [17 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca(NO3)2, 1.5 mM HEPES, pH 7.6] in an incubator with the same temperature and light conditions as the adult fish. At 1 dpf, embryos were manually dechorionated using Watchmaker’s forceps and screened for normal development before use in experiments. All animal care and experiments were conducted in accordance with protocols approved by the University of Massachusetts Amherst Institutional Animal Care and Use Committee (IACUC; Protocol Number 2019-0067). Animals were treated humanely with due consideration to the alleviation of stress and discomfort.

2.2. Chemicals

3,3′-dichloro-4-sulfooxy-biphenyl (4-PCB-11-Sulfate) was synthesized at the Iowa Superfund Research Program (Iowa City, IA) via the Suzuki coupling reaction as described elsewhere (Grimm et al. 2013; Lehmler and Robertson 2001). The authentication of this compound followed guidelines established previously (Li et al. 2018) and is documented elsewhere (Sethi et al. 2017). 4-PCB-11-Sulfate was dissolved in 100% dimethyl sulfoxide (DMSO) from Fisher Scientific (Fair Lawn, NJ). Serial dilutions were made, and all stock solutions were stored in glass amber vials at −20°C. Stocks were fully thawed and vortexed before use.

2.3. Chemical Exposures

The 0.2 μM concentration used in the experiments was chosen to mimic environmental and tissue concentrations observed in other studies (Grimm et al. 2017; Koh et al. 2015) and for comparison with our previous work (Roy et al. 2020). While this concentration is still higher than concentrations found in the environment, our previous work with PCB-11 has demonstrated that less than 1% of the original concentration is taken up into fish tissues (Roy et al. 2019), resulting in comparable concentrations to those detected in human samples (Grimm et al. 2017; Koh et al. 2015; Sethi et al. 2017).

At 1 dpf, dechorionated Nrf2a wildtype or mutant embryos were exposed in 5 mL of 0.3x Danieau’s media in 20 mL glass scintillation vials, with 5 embryos per vial and 4 vials per exposure group. Each vial was dosed with either DMSO or 0.2 μM 4-PCB-11-Sulfate, with a final DMSO concentration of 0.05% v/v across all vials. Each day from 2-4 dpf, 50% of the Danieau’s-based media solution was replaced. At 5 dpf, zebrafish were moved to individual 20 mL glass scintillation vials (1 fish per vial) containing 5 mL of the appropriate Danieau’s-based media solution. From 6-8 dpf, exposure group media were made based on system water obtained from the recirculating Aquaneering system housing the laboratory’s adult fish colony (controlled for pH, conductivity, and undergoes UV disinfection); 5 mL of the appropriate solution was added to each vial each day, so that at 8 dpf each vial contained 20 mL of solution. From 9-14 dpf, exposure group solutions were made in system water and 50% of the solution (10 mL) was replaced per vial. Fish were fed standard juvenile GEMMA Micro 75 (Skretting, Westbrook, ME) once per day starting at 5 dpf. This experiment was repeated 8 times for a total of 160 zebrafish per each of the 4 exposure groups. At 10 dpf, 2 of the experimental replicates were imaged and then returned to their individual vials. At 15 dpf, zebrafish from all experimental replicates were rinsed and then subsets of fish were imaged live or collected for RNAseq or fatty acid profiling.

2.4. Microscopy and Image Analysis

For all imaging, live larvae were sedated by a 10 second exposure to 2% v/v MS-222 solution (prepared as 4 mg/mL tricaine powder in water, pH buffered, and stored at −20°C until thawed for use) before being mounted individually in drops of 3% methylcellulose, with larvae in a right-lateral orientation for pancreas imaging. All imaging took place on a Zeiss Stereo Axio Zoom.V16 equipped with an HXP 200 C light source (Carl Zeiss Inc.). Whole organism brightfield images were captured at 20x and 16x total magnification for 10 dpf and 15 dpf, respectively, and pancreas images were captured at 100x total magnification. All images were blinded and then endpoints for body length and pancreatic islet area were measured with the Zen Lite program (Carl Zeiss Inc.). All measured outcomes were analyzed so that each fish was n=1 since fish in these experiments were raised in individual vials.

2.5. Fatty Acid Profiling

At 15 dpf, 5 fish from each exposure group were collected and pooled into a single Eppendorf tube for each of 6 experimental replicates to make n=6 per exposure group. Media in each tube was replaced with 100 μL nanopure water; tubes were placed on ice for 30s, sonicated with an Emerson Industrial Branson Sonifier® (Danbury, CT), and stored at −80°C until processing. Lipids in the homogenized samples were extracted by CHCl3:MeOH (2:1) (Folch et al. 1957) and methylated with methanolic HCl (1.5 M) as previously described (Park et al. 2001). Fatty-acid methyl esters were analyzed on a gas chromatography-mass spectrometer, GC-MS-QP2010 SE (Shimadzu, Japan), using a Supelcowax-10 column (100 m, 0.25 mm i.d., 0.25 μm film thickness, Sigma Aldrich, St Louis, MO, USA). Helium was used as the carrier gas and the column oven temperature started at 50°C, increased to 200°C at 20°C/min, then to 220°C at 2°C/min, and held at 220°C for 162.5 min. The injector was maintained at 220 °C (with split ratio of 1:1). For MS, the interface was 220°C and ion source was 200°C. An electron ionization (EI) with 70 eV with full scan mode in 35 to 500 m/z range with 0.3s of scan time were used. The fatty acid methyl esters were identified by comparing their retention times with the standards and confirmed by the National Institute of Standards and Technology (NIST) mass spectrum library. In total, 11 fatty acids were identified. The 4 saturated fatty acids analyzed were myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), and arachidic acid (C20:0). The 3 monounsaturated fatty acids (MUFAs) analyzed were palmitoleic acid (C16:1n-7), oleic acid (C18:1n-9), and vaccenic acid (C18:1n-7). The 4 polyunsaturated fatty acids (PUFAs) analyzed were linoleic acid (C18:2n-6), arachidonic acid (C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3), and docosahexaenoic acid (DHA, C22:6n-3). All other fatty acids not measured were classified together as “other” fatty acids. In addition to the zebrafish samples, zebrafish food (GEMMA Micro 75) was analyzed in triplicate for fatty acids. Data are reported as relative percentages of the total fatty acids.

2.6. RNA isolation, RNA library prep, and RNA sequencing (RNAseq)

At 15 dpf, 5 fish from each exposure group were collected and pooled into a single Eppendorf tube from each of 3 experimental replicates to make n=3 per exposure group. Tubes were placed in ice for 30s, media was replaced with 500 μL Trizol, and then tubes were stored at −80°C until processing. RNA was isolated using the RNA Clean Concentrate Kit with in-column DNase-I treatment (Zymo Research Corp.), following manufacturer instructions. The quantity of RNA was assayed on a Qubit using an RNA BR assay (Life Technologies Corp.), and the quality was assessed on an Agilent 2100 Bioanalyzer using an RNA 6000 Nano Assay (Agilent Technologies Inc). Total RNA was used to isolate poly(A) mRNA and libraries were prepared using the NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs) following the manufacturer instructions. The quantity of the library was assayed using a Qubit DNA HS assay (Life Technologies Corp.), and the quality was analyzed on a Bioanalyzer (Agilent Technologies Inc). Libraries were pooled and sequenced on an Illumina NextSeq 500 platform with 76 bp paired-end sequencing chemistry. Data was automatically uploaded to the Illumina Basespace platform for further processing. RNAseq data was also uploaded to the NCBI Gene Expression Omnibus (GEO) platform and can be accessed with the accession # PRJNA718696.

2.7. Bioinformatics

Raw fastq sequencing files were downloaded from Basespace and uploaded to the Galaxy web public server at usegalaxy.org to analyze the data (Afgan et al. 2018). The 4 Forward and 4 Reverse files for each of the 12 samples were concatenated into 1 Forward and 1 Reverse file per sample using the “Concatenate datasets tail-to-head (cat)” tool (Gruening 2014), and then the “fastp” tool was used for preprocessing quality control and trimming (Chen et al. 2018). The zebrafish genome GRCz11 was uploaded to Galaxy from ENSEMBL and was used for read alignment with the “HISAT2” tool. The “Infer Experiment” and the “Convert GTF to BED12” tools were used to ensure correct strand alignment, and the “MultiQC” tool was used to view mapping statistics. The “StringTie” tool was used to create gene count files, the “DESeq2” was used to run differential gene expression analyses, and the “Filter” tool was used to view the results for protein coding genes. The “Annotate DESeq2/DEXSeq output tables”, “Join two datasets”, and “Table Compute” tools were used to prepare files to generate a heatmap of differential gene expression using the “heatmap2” tool. The “Gene length and GC content” tool was used to generate files for the “goseq” tool, which was used for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis using the built-in GRCz10 zebrafish genome for the tool. The “Volcano Plot” and “Pathview” tools were also used to view differential gene expression and pathway analysis, respectively. Galaxy training tutorials were helpful in completing this analysis (Afgan et al. 2018; Batut et al. 2021).

2.8. Statistical Analyses

A Log-Rank test was performed for survival and a Wilcoxon nonparametric test was performed for secondary islet counts. All statistics were conducted with JMP® Pro software version 15.1.0 (Cary, NC). The unit of replication for 4 dpf experiments was the vial, containing up to 5 pooled larvae. The unit of replication for 15 dpf growth and pancreas endpoints was the individual fish, and the unit of replication for fatty acid profiling and RNAseq at 15 dpf was pooled groups of 5 fish with n=6 per exposure group for fatty acid profiling and n=3 per exposure group for RNAseq. Statistical significance was considered using a 95% confidence interval (α=0.05).

3. Results

3.1. 4-PCB-11-Sulfate exposures increase larval survival

A total of 8 experiments were conducted with 20 fish in each exposure group per experiment, and each fish was inspected daily for survival and recorded (Supplemental Table 1). We observed that out of 20 fish per experimental replicate, an average of 16.9 fish, or 85%, of Nrf2a wildtype fish and an average of 15.9 fish, or 80%, of Nrf2a mutant fish exposed to 0.2 μM 4-PCB-11-Sulfate survived to 15 dpf. This was significantly greater than the average of 14.6 fish, or 73%, of Nrf2a wildtype fish and the average of 13 fish, or 65%, of Nrf2a mutant fish exposed to the DMSO vehicle control that survived to 15 days (Figure 1).

Figure 1: Fish under chronic exposures from 1-15 dpf to 0.2 μM 4-PCB-11-Sulfate survive longer than untreated fish.

Figure 1:

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Zebrafish embryos were exposed daily between 1-15 dpf to 0.2 μM 4-PCB-11-Sulfate or DMSO and survival was checked every day. At 15 dpf fish exposed to 0.2 μM 4-PCB-11-Sulfate survived significantly better than fish exposed to DMSO (mean±SEM, n=8 experiments of 20 fish grown in individual vials from 5-15 dpf, Log-Rank test, p<0.05 at 15 days, W=wildtype, M=mutant, 4-PCB-11-SO4=4-PCB-11-Sulfate).

3.2. 4-PCB-11-Sulfate exposures increase body length and primary pancreatic islet area

Subsets of Nrf2a wildtype and mutant zebrafish exposed daily to DMSO or 0.2 μM 4-PCB-11-Sulfate were imaged at 10 dpf and 15 dpf to investigate exposure and genotype effects on growth and primary pancreatic islet development. At 10 dpf, independent of genotype, fish exposed to 0.2 μM 4-PCB-11-Sulfate were significantly longer by 4% (Figure 2A) and had significantly larger pancreatic islet area by 12% (Figure 2B) compared to unexposed fish. Genotype effects were not observed for length, but pancreatic islet area was dependent on genotype, with Nrf2a mutant fish experiencing a significant decrease in area by 7% compared to wildtype fish. No interaction effects were observed for these endpoints at this timepoint, and no differences were observed for the number of secondary islets (Figure 2C). Representative images of whole fish and islets can be seen in Figure 2D. At 15 dpf, the increases in length and pancreatic islet area for fish exposed to 0.2 μM 4-PCB-11-Sulfate persisted but the differences were slightly reduced. Independent of genotype, fish exposed to 0.2 μM 4-PCB-11-Sulfate were significantly longer by 3% (Figure 2E) and had significantly larger pancreatic islet area by 8% (Figure 2F) compared to unexposed fish. At 15 dpf, body length was dependent on genotype, with Nrf2a mutant fish significantly shorter by 4% compared to wildtype fish; genotype effects were not observed for pancreatic islet area. No interaction effects were observed for these endpoints at this timepoint, and no differences were observed between exposure groups for the number of secondary islets (Figure 2G). Representative images of whole fish and islets can be seen in Figure 2H.

Figure 2: Nrf2a is important for growth and 0.2 μM 4-PCB-11-Sulfate increases overall growth and pancreatic islet area independent of Nrf2a.

Figure 2:

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Zebrafish embryos were exposed daily between 1-15 dpf to 0.2 μM 4-PCB-11-Sulfate or DMSO in 8 experiments of 20 fish per exposure group. At 10 dpf 2 experiments were imaged for A) length, B) primary islet area, and C) secondary islet counts (n=33-39 fish per exposure group across 2 experiments, 2-Way ANOVA with Tukey’s post-hoc test for length and primary islet area, Wilcoxon non-parametric test for secondary islets, p<0.05). D) Representative images of whole fish and gut area showing islets at 10 dpf. At 15 dpf fish subsets of all 8 experiments were imaged for E) length, F) primary islet area, and G) secondary islet counts (n=54-88 fish per exposure group across 8 experiments, 2-Way ANOVA with Tukey’s post-hoc test for length and primary islet area, Wilcoxon non-parametric test for secondary islets, p<0.05). H) Representative images of whole fish and gut area showing islets at 15 dpf. * indicates a significant exposure effect compared to DMSO controls, † indicates a significant genotype effect, W=wildtype, M=mutant, 4-PCB-11-SO4=4-PCB-11-Sulfate.

3.3. 4-PCB-11-Sulfate exposures decrease Docosahexaenoic acid (DHA) in Nrf2a mutant larvae

At 15 dpf, fish from each exposure group were analyzed for 11 major fatty acids. Among all exposure groups, saturated fatty acids comprised 57-59%, monounsaturated fatty acids (MUFAs) 16-17%, polyunsaturated fatty acids (PUFAs) 16-20%, and other fatty acids 7-8% of total fatty acids (Figure 3A). Since more variability was observed for PUFAs, individual fatty acids within this group were examined more closely. Among the n-3 fatty acids, Nrf2a mutant fish exposed to 0.2 μM 4-PCB-11-Sulfate experienced a slight decrease in Eicosapentaenoic acid (EPA) (Figure 3B) and a significant decrease in DHA (Figure 3C) compared to Nrf2a wildtype fish exposed to 4-PCB-11-Sulfate, representing an interaction effect. Among the n-6 fatty acids, no 4-PCB-11-Sulfate exposure effects, Nrf2a genotype effects, or interaction effects were detected for arachidonic acid (Figure 3D) or linoleic acid (Figure 3E). Raw data for all 11 fatty acids from both zebrafish and fish food samples can be viewed in Supplemental Tables 2 and 3.

Figure 3: Nrf2a mutant fish exposed to 0.2 μM 4-PCB-11-Sulfate at 15 dpf have less DHA than Nrf2a wildtype fish exposed to 4-PCB-11-Sulfate.

Figure 3:

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Zebrafish embryos were exposed daily between 1-15 dpf to 0.2 μM 4-PCB-11-Sulfate or DMSO and at 15 dpf 5 embryos were pooled from each exposure group from 6 experiments. A) Fatty acid composition across saturated (C14:0, C16:0, C18:0, and C20:0), monounsaturated fatty acids (MUFAs C16:1n-7, C18:1n-9, and C18:1n-7), polyunsaturated fatty acids (PUFAs C18:2n-6, C20:4n-6, C20:5n-3, and C22:6n-3), and other fatty acids totaling 100% for each exposure group. PUFAs were examined closer and were compared between exposure groups for B) eicosapentaenoic acid (EPA), C) docosahexaenoic acid (DHA), D) arachidonic acid, and E) linoleic acid (n=6 of 5 pooled fish per exposure group across 6 experiments, 2-Way ANOVA with Tukey’s post-hoc test, p<0.05). # indicates a significant interaction effect, W=wildtype, M=mutant, 4-PCB-11-SO4=4-PCB-11-Sulfate.

3.4. Redox, drug, and fatty acid metabolism genes are decreased in Nrf2a mutant fish

At 15 dpf, 5 fish from each of the 4 exposure groups were pooled in 3 experimental replicates and underwent RNA sequencing. Raw fastq files were uploaded to the Galaxy web platform and the analysis pipeline and tools used can be viewed in Figure 4A. The DESeq2 tool was used to perform differential expression analysis of the 25,432 protein coding genes detected. When 4-PCB-11-Sulfate exposure was used as the primary factor and Nrf2a genotype as the secondary factor in the analysis, no differentially expressed genes (DEGs) with an adjusted p-value <0.05 were detected. Detected DEGs were dependent on genotype: when Nrf2a genotype was used as the primary factor alone, 338 DEGs were detected, and when 4-PCB-11-Sulfate exposure was added as the secondary factor in the analysis, 308 DEGs were detected with an adjusted p-value <0.05. Normalized gene counts for the 308 DEGs in each of the 12 samples were transformed to z-scores and are displayed in Figure 4B.

Figure 4: RNAseq pipeline and differential gene expression.

Figure 4:

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A) Galaxy pipeline of data processing and tools utilized (tool names are in parentheses). B) Z-score normalized differential gene expression for the 308 DEGs with an adjusted p-value <0.05 for the 12 samples across the 4 exposure groups. Blue colors indicate downregulation and red colors indicate upregulation.

The entire list of genes and their adjusted p-value <0.05 significance status with genotype as the primary factor and exposure as the secondary factor (308 DEGs) was submitted to the goseq tool in Galaxy for detection of enriched GO Molecular Function (MF), Biological Process (BP), and Cellular Component (CC) categories. We observed 49 categories significantly enriched with an adjusted p-value <0.05 (Supplemental Table 4). The top 10 categories are displayed in Figure 5A and reveal categories related to Nrf2a function such as oxidoreductase activity and oxidation-reduction process. The same list was submitted to goseq for KEGG pathway analysis, and we observed 5 KEGG pathways that were significantly enriched with adjusted p-values <0.05 and include glutathione metabolism, drug metabolism-cytochrome p450, metabolic pathways, metabolism of xenobiotics by cytochrome p450, and linoleic acid metabolism (Figure 5B). All DEGs for the KEGG Glutathione metabolism and KEGG linoleic acid metabolism pathways are displayed with their fold changes (Figure 5B).

Figure 5: RNAseq pathway analysis.

Figure 5:

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A) The top 10 significantly enriched GO categories of 50 categories with an adjusted p-value <0.05. B) All significantly enriched KEGG pathways with an adjusted p-value <0.05. DEGs and their log2 fold changes are shown for the glutathione metabolism and linoleic acid metabolism pathways.

4. Discussion

In this study, we investigated the role of Nrf2a on survival, pancreas development, fatty acid profiles, and gene expression at 15 dpf after exposures to 0.2 μM of the PCB-11 metabolite 4-PCB-11-Sulfate starting at 1 dpf. At 15 dpf, independent of genotype, fish exposed to 4-PCB-11-Sulfate survived significantly better at 85% and 80% for Nrf2a wildtype and mutant fish, respectively, compared to 73% and 65% survival for unexposed Nrf2a wildtype and mutant fish, respectively (Figure 1). Previous studies in our research group involving exposures to a different environmental chemical, perfluorooctanesulfonic acid (PFOS), in zebrafish out to 15 dpf noted >80% survival for all exposure groups, including the unexposed DMSO control group (Sant et al. 2021a). This previous study used 150 mL beakers from 5-15 dpf for individual fish, where the increased beaker space and media may have contributed to their increased baseline survival. In the precursor study to this current work (Roy et al. 2020), wildtype zebrafish were exposed from 1-15 dpf to either DMSO, the parent PCB-11 compound (0.2 μM), or either of two PCB-11 metabolites, 4-OH-PCB-11 or the 4-PCB-11-Sulfate used in this study (both at 0.2 μM), using the same exposure paradigm involving 20 mL vials. In that work, survival was also greater at 15 dpf in fish exposed to 0.2 μM 4-PCB-11-Sulfate at 89% compared to 75% for fish exposed to DMSO (data not previously reported). The 75% survival for unexposed wildtype fish in the previous work (n=60 over 3 experiments) is similar to the 73% survival for unexposed wildtype fish for this study. In both studies, DMSO was used as the vehicle for 4-PCB-11-Sulfate delivery and DMSO from the same stock and in the same concentration was delivered to the control fish. To our knowledge, no other study has reported increased larvae survival after a chronic low concentration exposure to a PCB. It remains unclear why exposure to 4-PCB-11-Sulfate increased survival for both experiments at 15 dpf and whether this is a compensatory effect in the short-term or whether this survival would persist at later time points. However, adaptive responses to toxicants usually have associated fitness costs, as seen with killifish from the Elizabeth River that are resistant to PCBs and PAHs (Clark and Di Giulio 2012; Meyer and Di Giulio 2003).

In addition to survival, fish that were exposed to 4-PCB-11-Sulfate had significantly larger primary pancreatic islets at both 10 and 15 dpf compared to unexposed fish (Figure 2B and 2F). To our knowledge, exposure to environmental pollutants have mostly resulted in decreases in pancreatic islet size, such as studies conducted with PFOS by others with juvenile stage mice (Qin et al. 2022) and by our group with PFOS and perfluorobutanesulfonic acid (PFBS) in early larval stage zebrafish up to 7 dpf (Sant et al. 2017; Sant et al. 2016; Sant et al. 2019), although we have also noted different outcomes depending on the chemical exposure and time point. For instance, developmental exposure to butylparaben significantly increased islet area whereas exposures to mono-2-ethylhexyl phthalate (MEHP) significantly decreased islet area, both studies with timepoints ending at 7 dpf or less (Brown et al. 2018; Jacobs et al. 2018). However, in another study exploring the effects of PFOS at later time points, the results showed at both 15 dpf and 30 dpf that PFOS significantly increased primary pancreatic islet area and volume and revealed that these increases corresponded to significantly “looser” islet structures and less islet sphericity (Sant et al. 2021a). The microscopy work undertaken for this current study was not able to measure volume, structure, or sphericity to the granularity investigated by Sant et al., but these results showed how an environmental contaminant like 4-PCB-11-Sulfate can influence pancreatic islet development.

Primary islet and overall pancreas morphology have been studied by many research groups, mostly in relation to diabetes disease onset and progression. In humans, individuals with either Type 1 or Type 2 diabetes have been characterized as having both reduced pancreatic islet mass as well as reduced overall pancreas mass, particularly for Type 1 diabetes patients (Yagihashi 2017). While diabetes was not specifically investigated in this work, pancreas size appears important for its ability to regulate blood glucose, though overall mass may not directly indicate functional ability, as characterized by Seiron et al. who showed from adult human Type 1 diabetes patient biopsies that pancreatic islet size was maintained despite a reduction in functional beta cells (Seiron et al. 2019). To add to the complexity of the variety of pancreas responses, a study in mice showed that pancreatic islets increased in β-cell mass, proliferation, density, and size during early stages of Type 2 diabetes onset, demonstrating hypertrophy and hyperplasia, with glucose tolerance tests indicating early beta cell dysfunction (Asghar et al. 2006). It is unclear whether the increased islet sizes from exposure to 4-PCB-11-Sulfate presented in this study represent looser structures as shown by Sant et al., represent hypertrophy and hyperplasia as shown by Asghar et al., or represent another response such as inflammation – an adaptive response of pre-diabetes in humans (Luc et al. 2019) – but any of these cases is indicative of abnormal development and dysfunction of the pancreas.

Although the survival and pancreatic islet effects we observed were independent of Nrf2a genotype, the effects observed for overall growth, fatty acid profiles, and in the RNAseq data were largely dependent on Nrf2a genotype. We observed that Nrf2a mutant fish at 15 dpf were 3-5% shorter than their wildtype counterparts, which is consistent with observations in separate experiments of Nrf2a wildtype and mutant fish at 4 dpf (Supplemental Methods 1 and Supplemental Figures 1 and 2), a previous study from our lab (Sant et al. 2021b), and is consistent with an overview of zebrafish Nrf family members at early developmental stages (Williams et al. 2013). Additionally, Nrf2a has been shown to be especially important during toxicant exposures to counter increases in reactive oxygen species that might occur. For instance, a previous study with Nrf2a mutant fish exposed to the dioxin-like toxicant PCB-126 experienced significantly increased aryl hydrocarbon receptor (Ahr) pathway dependent hepatic Cyp1a induction and increased deformities compared to wildtype fish exposed to PCB-126 (Rousseau et al. 2015); Supplemental Figure 3 confirms these results for PCB-126 exposures in Nrf2a wildtype and mutant fish. In previous work, 20 μM exposures to PCB-11 and 4-PCB-11-Sulfate have also demonstrated their ability to inhibit Cyp1a in wildtype fish at 4 dpf, resulting in increased toxicity (Roy et al. 2020; Roy et al. 2019). In this current study, these exposures were repeated with Nrf2a wildtype and mutant fish for 1-4 dpf exposures (Supplemental Figures 1-3), though the toxicity observed for Nrf2a mutant fish exposed to PCB-11 or 4-PCB-11-Sulfate was not significantly more than the toxicity incurred by Nrf2a wildtype fish.

In the fatty acid profiling, Nrf2a did not play a role in any of the 11 fatty acids analyzed, however, an interaction effect was observed where Nrf2a mutant fish exposed to 4-PCB-11-Sulfate experienced a significant decrease in the omega-3 fatty acid DHA (Figure 6C). Though variations in the relative percentages of individual fatty acids fluctuated across the 6 experimental replicates (Supplemental Table 2), the decreases observed for DHA were consistent. These decreases were also interesting considering that the relative percentage of DHA in the fish food (Supplemental Table 3) is much less than the DHA measured in the fish, perhaps pointing to disrupted upstream or downstream metabolism processes. DHA is an important fatty acid for brain function and cognitive development early in life, and may play a role in preventing neurodegenerative disorders (Balakrishnan et al. 2021). DHA has also been shown to activate Nrf2 to maintain redox balance and confer neuroprotection upon toxicant exposures to agrochemicals (Drolet et al. 2021). PCB-11 has already been identified as a threat to the developing brain via dendritic growth and arborization through cAMP response element-binding protein (CREB) signaling (Klocke et al. 2020; Sethi et al. 2018), a transcription factor important for neurogenesis and cognitive development. Interestingly, it has been shown that DHA treatment also activates CREB to confer neuroprotection (Figueroa et al. 2012), including protection of the hippocampus associated with diabetes (“diabetic encephalopathy”) through reducing oxidative stress, inflammation, and apoptosis (Alvarez-Nölting et al. 2012). The results from our current study indicate that Nrf2a may be protective of DHA metabolism, especially in the presence of environmental chemicals like 4-PCB-11-Sulfate.

Environmental chemicals can disrupt the regulation of genes that govern the biological processes described above such as fatty acid metabolism, pancreas growth, and the response to oxidative stress (Brown et al. 2018; Jacobs et al. 2018; Sant et al. 2021b; Timme-Laragy et al. 2015). The RNAseq analysis of 15 dpf Nrf2a wildtype and mutant fish exposed to DMSO or 4-PCB-11-Sulfate detected 308 differentially expressed genes (DEGs) with an adjusted p-value <0.05 (Figure 4). However, many of these DEGs were more dependent on Nrf2a genotype than 4-PCB-11-Sulfate exposure and included several downregulated glutathione S-transferase (GST) genes, which are responsible for conjugating reduced glutathione to a variety of electrophilic substances for biotransformation. Specifically in this study, GST pi 1 (gstp1, −0.67 log2 fold change), GST omega 1 (gsto1, −0.49 log2 fold change), GST theta 1a (gstt1a, −0.34 log2 fold change), GST rho (gstr, −0.24 log2 fold change), and microsomal GST 3a (mgst3a, −0.27 log2 fold change) were all downregulated. The enzyme responsible for catalyzing oxidized glutathione into its reduced form, glutathione reductase (gsr, −0.37 log2 fold change), and an enzyme responsible in glutathione synthesis, glutamate-cysteine ligase, catalytic subunit (gclc, −0.25 log2 fold change), were also downregulated. An antioxidant enzyme responsible for the removal of reactive oxygen species, superoxide dismutase 2, (sod2, 0.61 log2 fold change), was upregulated.

When the results of the DEG analysis were submitted for gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, most of the significantly enriched GO categories and KEGG pathways in the output were related to redox balance (Figure 5). However, some GO categories close to the 0.05 cutoff seemed to indicate the influence of 4-PCB-11-Sulfate exposure. For instance, just after the adjusted p-value 0.05 cutoff, categories such as “sulfur compound metabolic process”, “response to toxic substance”, “drug catabolic process”, “response to xenobiotic stimulus”, and “benzene-containing compound metabolic process”, all with an adjusted p-value <0.1, were identified (Supplemental Table 4). In addition, one of the significantly enriched KEGG pathways was the linoleic acid metabolism pathway, which includes 3 DEGs from our analysis, cyp2p10, cyp2j20, and cyp2p9, all downregulated and potentially related to the increased hepatic lipid accumulation that we observed previously (Roy et al. 2020). Both zebrafish cyp2j20 and cyp2p10 genes are orthologous to the human CYP2J2 gene, which is important in fatty acid metabolism (Furge and Guengerich 2006). CYP2J2 has also been identified as a target to reduce inflammation, oxidative stress, and in attenuating disease phenotypes, including obesity and non-alcoholic fatty liver disease (NAFLD) (Abraham et al. 2014; Chen et al. 2015).

Studies by another group have shown that exposure to PCB-11 and 4-PCB-11-Sulfate in rodents is primarily distributed to the liver and adipose tissue (Grimm et al. 2015; Zhang et al. 2021), and in our previous work, we observed that 4-PCB-11-Sulfate significantly increased hepatic neutral lipids in zebrafish at 15 dpf (Roy et al. 2020). The liver and the pancreas communicate with each other to regulate blood glucose levels and hepatic storage via pancreatic insulin-producing β-cells and glucagon-producing α-cells, among other important signaling molecules. We did not detect DEGs that might explain the increased pancreatic islet area we observed. For instance, no significant changes in gene expression were detected for pancreatic and duodenal homeobox 1 (pdx1), pancreas associated transcription factor 1a (ptf1a), preproinsulin (ins), either of the insulin receptors (insra and insrb), or glucagon (gcga). However, the brain isoform of glycogen phosphorylase (pygb, −0.28 log2 fold change) was significantly downregulated. Interestingly, the human ortholog PYGB has been associated with the acute insulin response to glucose (Rich et al. 2009) and has been identified as a therapeutic target for the prevention of various tumor progressions, including hepatocellular carcinoma (Cui et al. 2020). Glucagon plays a key role in the liver-pancreas axis, regulating amino acid metabolism (Winther-Sørensen et al. 2020), and in human patients with fatty liver disease where glucagon signaling is disrupted, has been shown to result in insulin resistance and hyperglucagonemia (Pedersen et al. 2020). Additionally, out of the 10 solute carrier (SLC) DEGs detected, SLC 2a1a (slc2a1a, 0.59 log2 fold change, orthologous to human SLC2A1) and SLC 2a2 (slc2a2, −0.34 log2 fold change, orthologous to human SLC2A2) are important for glucose transport activity and are implicated in Type 2 diabetes (Sander et al. 2019). Figure 6 captures some of the connections observed in this current study and observed in previous studies by our group and others. Follow-up research that presents glucose challenges to the fish in this study would be beneficial to understand the physiological responses of the islet area and gene expression results that were observed.

Figure 6: Summary figure.

Figure 6:

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Observations and connections from factors of interest PCB-11, 4-PCB-11-Sulfate, and lack of functional nrf2a in zebrafish organized by whole body, organ, or cellular level. Small width arrows connect these factors of interest to outcomes observed in this study (no references on the arrow) and in previous studies (references on the arrows). Larger width arrows indicate the direction of observed change. Ahr = Aryl hydrocarbon receptor; BNF = beta naphthoflavone; SF = supplemental figure.

Overall, this study contributes to the increasing literature on environmental contaminants that can potentially disrupt fatty acid synthesis, lipid metabolism, and enzymes important for clearing xenobiotic substances. Specifically, this study examined the role of Nrf2a on development and in the context of developmental exposures to a prevalent non-ortho-substituted lower-chlorinated PCB congener. We observed that both 4-PCB-11-Sulfate and Nrf2a can affect body growth and pancreatic islet area, and that Nrf2a affects the omega-3 fatty acid DHA under 4-PCB-11-Sulfate exposures. In addition, the results of the RNAseq analysis point to the role of Nrf2a in maintaining redox balance, but also in fatty acid metabolism and glucose transport. Though the samples collected for RNAseq were of whole pooled fish, future work that is able to collect single-cell or isolated hepatic or pancreatic tissue might be more beneficial for understanding the effects of Nrf2a and 4-PCB-11-Sulfate on these organs.

5. Conclusions

The influence of Nrf2a on survival, pancreas development, fatty acid profiles, and gene expression was investigated in the zebrafish embryo model in the context of exposures to 4-PCB-11-Sulfate between 1-15 dpf. We observed genotype dependent effects at 15 dpf where Nrf2a appeared important for growth and for DHA synthesis in the presence of 4-PCB-11-Sulfate. Fish independent of genotype that were exposed to 4-PCB-11-Sulfate survived significantly better and had 12% larger islets at 10 dpf and 8% larger islets at 15 dpf. The underlying reason behind these genotype dependent and independent effects remain unclear, though perhaps are in alignment with symptoms of early onset of diabetes. Several differentially expressed genes with adjusted p-values <0.05 were identified that are involved in fatty acid oxidation, glucose transport, and glucagon signaling. Further studies that examine adulthood effects of Nrf2a in the context of early developmental environmental chemical exposure would be beneficial.

Supplementary Material

supplementary data

Highlights.

  • Nrf2a is important for growth during larval and juvenile development

  • Nrf2a is important for DHA production under 4-PCB-11-Sulfate exposures

  • 4-PCB-11-Sulfate increases survival and pancreatic islet area in juvenile fish

  • Genes related to glutathione, xenobiotic metabolism, and fatty acid metabolism are downregulated in Nrf2a mutant fish

Acknowledgments

We would like to thank members of the Timme-Laragy laboratory for providing excellent zebrafish care at UMass Amherst. We would also like to thank Dr. Hans-Joachim Lehmler, Dr. Keri Hornbuckle, and Dr. Xueshu Li from the Iowa Superfund Research Program at the University of Iowa for the synthesis and sharing of the chemicals used in this study. We would also like to thank Dr. Ravi Ranjan from the UMass Amherst Genomics Resource Laboratory and Dr. Andrew McArthur from McMaster University for helpful conversations related to RNAseq data processing.

Funding

Funding for this work was provided in part by the National Institutes of Health (NIH) (grant number R01 ES025748 to ART-L) and the Iowa Superfund Research Program (ISRP) of the National Institute of Environmental Health Sciences (grant number P42 ES013661) for PCB compound synthesis. Funding was also provided to MAR through the NIH T32 Biotechnology Training Program at the University of Massachusetts Amherst (National Research Service Award T32 GM108556) and through an NIH F31 predoctoral fellowship (National Research Service Award F31 ES030975).

Abbreviations

ANOVA

analysis of variance

DHA

docosahexaenoic acid

DMSO

dimethyl sulfoxide

dpf

days post fertilization

GSH

reduced glutathione

GSSG

oxidized glutathione

GST

glutathione S-transferase

Nrf2a

nuclear factor erythroid 2-related factor 2

PCB

polychlorinated biphenyl

PCB-11

3,3’-dichlorobiphenyl

4-PCB-11-Sulfate

3,3′-dichloro-4-sulfooxy-biphenyl

RNAseq

RNA sequencing

Footnotes

Declaration of Competing Interest

The authors have no competing interests to declare.

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