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. Author manuscript; available in PMC: 2023 Jun 21.
Published in final edited form as: Birth Defects Res. 2022 Dec 5;115(4):458–473. doi: 10.1002/bdr2.2132

Tris(4-chlorophenyl)methane (TCPM) and tris(4-chlorophenyl)methanol (TCPMOH) disrupt pancreatic organogenesis and gene expression in zebrafish embryos

Peyton W Wilson 1, Christine Cho 1, Nicholas Allsing 1, Saleha Khanum 1, Pria Bose 1, Ava Grubschmidt 1, Karilyn E Sant 1,*
PMCID: PMC10284116  NIHMSID: NIHMS1905680  PMID: 36470842

Abstract

Tris(4-chlorophenyl) methane (TCPM) and tris(4-chlorophenyl)methanol (TCPMOH) are anthropogenic environmental contaminants believed to be manufacturing byproducts of the organochlorine pesticide dichlorodiphenyltrichloroethane (DDT) due to environmental co-occurrence. TCPM and TCPMOH are persistent, bioaccumulate in the environment, and are detected in human breast milk and adipose tissues. Due to the toxicity of similar organochlorines, we determined impacts on zebrafish pancreatic growth and gene expression following developmental exposures. Zebrafish embryos were exposed to 50 nM TCPM or TCPMOH beginning at 24 hours post fertilization (hpf) and exposures were refreshed daily. At 96 hpf, pancreatic growth and islet area were directly visualized in Tg(ptf1a::GFP) and Tg(insulin::GFP) embryos, respectively, using microscopy. Gene expression was assessed at 100 hpf with RNA sequencing. Islet and total pancreas area were reduced by 20.8% and 13% in embryos exposed to 50 nM TCPMOH compared to controls. TCPM did not induce significant morphological changes to the developing pancreas, indicating TCPMOH, but not TCPM, impairs pancreatic development despite similarity in molecular responses. Transcriptomic responses to TCPM and TCPMOH were correlated (R2=0.903), and pathway analysis found downregulation of processes including retinol metabolism, circadian rhythm, and steroid biosynthesis. Overall, our data suggest that TCPM and TCPMOH may be hazardous to embryonic growth and development.

Keywords: pancreatic development, embryo, organogenesis, tris(4-chlorophenyl)methane, TCPM

1. INTRODUCTION

Tris(4-chlorophenyl)methane (TCPM) and tris(4-chlorophenyl)methanol (TCPMOH) are emerging organochlorine contaminants of concern (Trego et al., 2019). TCPM and TCPMOH are structurally related to the organochlorine pesticide Dichlorodiphenyltrichloroethane (DDT), and evidence suggests they are likely manufacturing byproducts of this legacy contaminant (Buser, 1995; de Boer, 1997). Concentrations of TCPM and TCPMOH in ecological and human samples are frequently significantly correlated with DDT concentrations, further corroborating the relation of these compounds (Falandysz et al., 1999; Kajiwara et al., 2001; Kivenson et al., 2019; Kunisue et al., 2004; Lebeuf et al., 2001).

TCPMOH was first identified in harbor seals from the Puget Sound in 1989 and has since been frequently detected with TCPM in various sediments, cetaceans, pinnipeds, coastal bird eggs, and other animals globally (de Boer, 1997; Falandysz et al., 1999; Jarman et al., 1992; Kajiwara et al., 2001; Kannan et al., 2004; Lebeuf et al., 2001; Millow et al., 2015; Shaul et al., 2015; Watanabe et al., 1999). Transplacental transfer of TCPMOH and TCPM has been confirmed in whales (Kajiwara, Kamikawa, et al., 2008) and maternal transfer of TCPMOH has been observed during seal gestation and lactation (Kajiwara, Watanabe, et al., 2008). Age dependent accumulation of TCPM and TCMOH has been observed in human adipose tissues and multiple studies have detected TCPM and TCPMOH in human breast milk, suggesting human exposure and potential maternal transfer of these compounds in humans (Kunisue et al., 2004; Minh et al., 2000; Rahman et al., 1993). Despite the extensive and widespread occurrences of TCPM and TCPMOH in ecological and human samples, and the potential for developmental exposure to humans through maternal transfer, few studies have characterized the developmental toxicity of these contaminants.

Studies characterizing the mechanisms of toxicity for TCPM and TCPMOH are limited, though several have suggested exposure to TCPMOH induces endocrine activity in mammals via antiandrogenic activity and changes to steroid hormone metabolism (Foster et al., 1999; Körner et al., 2004; Schrader & Cooke, 2002; Segura-Aguilar et al., 1997). However, in vitro studies suggest TCPMOH and TCPM act as estrogen mimics (Lascombe et al., 2000). In rats, TCPMOH was found to induce Phase I biotransformation enzymes, mainly cytochrome p450s, and phase II microsomal enzyme activities, mainly glutathione-S-transferases (Poon et al., 1997). In gene expression analysis of dolphin blubber from the Southern California Bight, increasing loads of TCPMOH corresponded with upregulating of CYP1b1 (Trego et al., 2019). These responses to TCPMOH indicate a xenobiotic response involving some genes involved in the aryl hydrocarbon receptor (AHR) and nuclear factor erythroid 2–related factor 2 (Nrf2) pathways. In zebrafish, embryonic mortality and developmental deformities increased in a dose-dependent manner after supraenvironmental exposures to TCPMOH. Gene expression of Cyp1a was impaired by acute exposure (4 h) to 50 nM TCPMOH in these zebrafish embryos (Navarrete et al., 2021).

Exposures to toxic chemicals during sensitive time periods, such as embryonic development, induce biochemical, molecular, cellular, or morphological responses that may lead to disease or metabolic dysfunction later in life (Sant, Jacobs, et al., 2017). Given the conserved development of the pancreas and the many shared developmental signaling pathways between humans and zebrafish, zebrafish exposures to toxicants may result in similar effects in humans (Sant et al., 2016). During zebrafish embryogenesis, two anlages emerge and fuse to form the endocrine and exocrine pancreas (Sant, Jacobs, et al., 2017). Endocrine tissue, located in the Islets of Langerhans and consisting mostly of insulin-producing beta cells, produces hormones which maintain glucose homeostasis (Sant, Jacobs, et al., 2017). Endocrine Islets are embedded in exocrine pancreas tissue, which produces and delivers digestive enzymes to the intestinal tract (Sant, Jacobs, et al., 2017). The pancreas is an especially sensitive target to toxic exposures due to low xenobiotic metabolism and low antioxidant capacity in pancreatic beta cells. Additionally, the pancreas has a high perfusion rate, which stimulates proficient drug or toxicant delivery to these sensitive tissues (Sant et al., 2016).

Following toxic exposures during organogenesis, structural deviations of the pancreas and altered gene expression have been observed. In zebrafish exposed to perfluorooctanesulfonic acid (PFOS), reduced beta cell area and pancreas length occurred with reduced gene expression of digestive enzymes and hormones governing glucoregulation (Sant, Jacobs, et al., 2017). Similar structural and transcriptional changes have been observed for other chemicals with endocrine activity, such as phthalates, perfluoroalkyl substances, parabens, and polychlorinated biphenyls. Here we aim to investigate the impact of embryonic exposures to the environmental contaminants TCPM and TCPMOH on the zebrafish developing pancreas. We hypothesize that exposure to TCPM and TCPMOH will disrupt organogenesis and function of the sensitive developing pancreas and induce changes to gene expression.

2. METHODS

2.1. Chemicals

Tris(4-chlorophenyl)methane (TCPM; CAS# 27575-78-6) was purchased from Matrix Scientific (Elgin, SC) and Tris(4-chlorophenyl)methanol (TCPMOH; CAS #3010-80-8) was purchased from Sigma Aldrich (St. Louis, MO). Dimethyl Sulfoxide (DMSO) was purchased from Fisher Scientific (Pittsburgh, PA). Concentrated stock (10,000X) solutions of TCPM [500 μM] and TCPMOH [500 μM] for embryonic exposures were prepared in DMSO and stored at −20 °C in amber glass vials away from light until use. Appropriate safety precautions were used during experimental procedures involving TCPM and TCPMOH, and all waste was disposed of in accordance with appropriate hazardous waste guidelines.

2.2. Animals & Husbandry

Wild-type (AB) strain zebrafish were originally obtained from the University of California San Diego. Transgenic zebrafish of the Tg(insulin::GFP) and Tg(ptf1a::GFP) strains were originally obtained from the University of Massachusetts Amherst as heterozygous populations. The Tg(insulin::GFP) strain was originally produced by Dr. Philip diIorio, and express GFP in insulin-producing pancreatic beta cells (diIorio et al., 2002). The Tg(ptf1a::GFP) strain was originally produced by Dr. Steven Leach, and express GFP throughout the exocrine pancreas, as well as the retina and parts of the brain (Godinho et al., 2005; Lin et al., 2004). All strains have been maintained as breeding colonies at San Diego State University since 2019, and are outcrossed every 4-5 generations onto an AB background reduce inbreeding.

Breeding colonies of fish used in these experiments were maintained at 28.5°C under a 12 h light:12 h dark photoperiod cycle in a recirculating Aquaneering zebrafish system at San Diego State University. Conductivity was maintained at 650-700, pH 7.2-7.3, and nitrates, nitrites, ammonia, and chlorine were monitored weekly. Fish were fed daily with the recommended amount of GEMMA Micro 300 powdered diet (Skretting; Westbrook, ME). Breeding populations were housed in tanks of approximately 15-20 adult fish with a 2:3 male: female ratio. was All procedures were reviewed and approved by the San Diego State University Institutional Animal Care and Use Committee (APF#: 21-08-007S) and Zebrafish were maintained in accordance with the requirements pertaining to animal subjects protections within the Public Health Service Policy and USDA Animal Welfare Regulations.

Embryos were collected from breeding tanks within 1 hour post fertilization (hpf), washed, and placed in clean polystyrene petri dishes containing 0.3X Danieau’s medium (17 mM NaCl, 2 mM KCl, 0.12 mM MgSO4, 1.8 mM Ca (NO3)2, 1.5 mM HEPES, pH 7.6). Collected embryos were screened for quality and viability and were incubated at 28.5°C on a 12 h light: 12 h dark photoperiod cycle. Clutches in which fertilization rates <80% or mortality prior to 24 hpf was >10% were excluded from experiments. Mortality or structural abnormalities in control embryos were screened throughout experiments, and no clutches violated quality control measures.

2.3. Exposures

Prior to all exposures, embryos were manually dechorionated at 24 hpf with watch-makers’ forceps. For microscopy experiments, Tg(insulin::GFP) or Tg(ptf1a::GFP) embryos were randomized across clutches and transferred to glass scintillation vials at a density of 5 embryos per vial. Each vial was prepared with 10 mL of 0.3X Danieau’s medium and 1 μL of a working stock to attain concentrations of 50 nM TCPM, 50 nM TCPMOH, or 0.01% v/v DMSO (vehicle control). These environmentally- and biologically-relevant concentrations were determined to fall below the NOAEL for acute developmental toxicity of TCPMOH in zebrafish embryos, so no mortality or gross embryonic defects were expected (Navarrete et al., 2021). Exposure media was 95% removed and refreshed daily on 2-3 days post fertilization (dpf). Microscopy was performed at 4 dpf, repeated 8 times for Tg(insulin:GFP) embryos and 10 times for Tg(ptf1a:GFP) embryos with 5-10 embryos in each exposure group per experiment to meet statistical power (β=0.2) requirements.

For RNA sequencing experiments, wild-type (AB) embryos were dechorionated and transferred to clean polystyrene petri dishes at 24 hpf with 12-35 embryos per dish. Each petri dish was prepared with 30 mL of 0.3X Danieau’s medium and 1μL of a working stock to attain concentrations of 50 nM TCPM, 50 nM TCPMOH, or 0.01% v/v DMSO. At 4 dpf, embryos were rinsed thoroughly, pooled, and collected for RNA isolation. Each sample contained 8-10 embryos, and experiments were replicated 5 times to generate 5 samples per exposure group. Samples were preserved in RNAlater solution (Fisher Scientific) and stored at −20°C until RNA isolation.

2.4. Microscopy

Tg(insulin:GFP) and Tg(ptf1a:GFP) were imaged at 4 dpf to observe fish length, islet and exocrine pancreas area, pancreas length, morphology, and yolk utilization. A Nikon Ti-2 inverted epifluorescent microscope was used to perform all imaging. Embryos were washed and briefly anaesthetized in 2% v/v MS-222 solution (prepared as 4 mg/mL Finquel tricaine powder in water, pH buffered, and stored at −20°C). Embryos were individually mounted in drops of 3% methylcellulose and in a right lateral orientation. Brightfield images were taken at 20X and 40X magnification to observe whole embryo development and measure fish length and yolk utilization. GFP images were taken at 100X magnification to observe pancreatic development and measure islet area, exocrine pancreas area, and pancreas length.

Nikon NIS Elements Advanced Research software was used to quantify measurements from images. Islet area and exocrine pancreas area were measured by capturing the perimeter of GFP expression. Pancreas length was measured linearly from the islet to the end of the pancreas. Rostral-to-caudal fish length was measured from brightfield images to quantify impacts to embryonic growth. Yolk utilization, an indicator of nutrient utilization and metabolic function, was measured by tracing the outline of the yolk sac. Hypermorphic (>90th percentile of controls) and hypomorphic (<10th percentile of controls) islet areas and pancreas lengths were recorded.

2.5. RNA Isolation & Sequencing

RNA sequencing was performed to measure changes in gene expression in embryos exposed to 50 nM TCPM or 50 nM TCPMOH. Following the exposure described above, pools of 8-10 4 dpf embryos were transferred from petri dishes and stored in RNAlater at −20°C until isolation. Immediately prior to RNA isolation, samples were thawed on ice and RNA later was removed. Samples were then pulse sonicated at 10% amplification using a Branson SFX250 Sonifer and RNA was isolated using the Thermo GeneJET RNA Purification Kit (Fisher Scientific) according to protocols. Extracted RNA was stored at −80°C until use.

Library preparation and RNA sequencing were performed at the University of California San Diego Institute for Genomic Medicine Genomics Center (San Diego, CA). An Agilent 4200 TapeStation was used to assess RNA quality and quantity (Agilent Technologies, Santa Clara, California). RNA integrity numbers (RINs) ranged from 8.6-9.8 and averaged 9.6 The Illumina Stranded mRNA Prep Kit was used according to manufacturer protocols with Poly(A) enrichment to prepare stranded libraries. Libraries were then sequenced using the Illumina NovaSeq 6000 platform using paired end 100 bp (PE100; 2x100bp) reads to a sequencing depth of 25 M reads.

2.6. Bioinformatics

FASTQ files were labeled according to exposure group and imported on a local instance of Galaxy (Afgan et al., 2018). Any low quality sequences were identified with FastQC (Andrews, 2010). Unwanted sequences were trimmed and filtered out with Cutadapt, in accordance with parameters set for paired-end reads, a quality cutoff of 20, and a minimum filter length of 20 (Martin, 2011). A zebrafish genome file and transcriptome annotation file, from the Lawson Lab at UMASS Medical School (v. 4.3.2), were aligned and annotated using Spliced Transcripts Alignment to a Reference (STAR) software (Dobin et al., 2013; Lawson et al., 2020). Using the output from STAR, reads per annotated gene were counted using the featureCounts program (Liao et al., 2014). Differentially expressed features between exposure groups were identified using DESeq2 software (Love et al., 2014). Genes were considered significantly changed if the DESeq FDR value was <0.05. Biologically relevant gene sets were identified by uploading the DRSeq2 output to the RNA-Enrich version of LRpath and using the KEGG Pathway database (Kim et al., 2012; Lee et al., 2015). FishEnrichR was used to perform Gene Ontology (GO) enrichment analysis (Chen et al., 2014; Kuleshov et al., 2016). Differentially expressed genes and pathway enrichment were considered significant using the Benjamini-Hochberg adjusted p-value (p<0.05). Raw data files and full DESeq2 results may be accessed as a complete series in the NCBI Gene Expression Omnibus (GSE198106).

2.7. Statistical Analyses

All data was recorded in Microsoft Excel, and all data was analyzed using IBM SPSS Statistics v28. To determine the appropriate statistical tests, Shapiro-Wilk tests and Levene’s tests were used to assess normality and variances, respectively. All data was normally distributed data and is presented as the mean ± standard error. ANOVA with Tukey (equal variances) or Games-Howell (unequal variances) post hoc tests were performed to compare means of continuous data. Chi-square tests were performed to compare frequencies of hypermorphic or hypomorphic pancreas morphologies. Confidence levels of 95% (α=0.05) were used for all experiments.

3. Results

3.1. Islet Development

Islet development was quantified by measuring the cross-sectional area of fluorescent beta cells in Tg(insulin:GFP) embryos imaged at 4 dpf (Figure 1A). The mean islet area was significantly reduced (p=0.042) in embryos exposed to TCPMOH compared to controls, though TCPM exposure did not significantly impact islet area (p=0.714). Mean islet area of embryos exposed to 50 nM TCPMOH and 50 nM TCPM were reduced by 20.8% and 6.8%, respectively. We also assessed incidence of hypomorphic and hypermorphic islet areas, defined as islet areas falling below the 10th percentile or above the 90th percentile of control islet areas (Figure 1B). Incidence of hypomorphic islets was elevated to 30% by TCPMOH exposures, but this increased incidence was not statistically significant compared to controls (p=0.117).

Figure 1. Islet Area.

Figure 1.

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Tg(insulin:GFP) Zebrafish embryos were imaged and measured at 96 hpf following exposure to 50 nM TCPMOH, 50 nM TCPM, or 0.01% DMSO. Asterisks * designate statistically significant changes from controls (p<0.05). A) The area of fluorescent beta cells in the primary islet is presented in a bar graph as the Mean +/− Standard Error. An AVOVA with a Tukey (equal variances) post-hoc test was used to compare means. Mean Islet Area of embryos exposed to 50 nM TCPMOH and 50 nM TCPM were reduced by 20.8% and 6.8%, respectively, compared to controls. B) Chi-square tests were used to compare incidence of hypomorphic and hypermorphic islet areas in each treatment group. Results are presented in a stacked bar graph, with the percent of islets that fell above the 90th percentile represented by gray bars (hypermorphic) and the percent of islets that fell below the 10th percentile represented by black bars (hypomorphic). The incidence of hypermorphic or hypomorphic islets was not statistically significant. n=20-29 embryos per treatment group.

3.2. Exocrine Pancreas Development

Exocrine pancreas development was assessed by measuring the cross-sectional area and length of the pancreas in Tg(ptf1a:GFP) embryos at 4 dpf. Exposure to 50 nM TCPMOH significantly reduced pancreas area by 13% (p=0.050) (Figure 2A). TCPM exposure reduced mean pancreas area by 8.8% but was not statistically significant (p=0.281). Incidence of hypomorphic pancreas areas due to TCPMOH exposure was increased to 30% (p=0.046) (Figure 2B). Pancreas length was measured as the length from the center of the primary islet to the posterior end of the pancreas, and is indicative of pancreatic extension during late organogenesis. No statistically significant differences in mean pancreas length were observed due to exposures (p>0.05) (Figure 3A). However, incidence of both hypermorphic and hypomorphic pancreas lengths was significantly elevated in the TCPMOH group (p=0.046) (Figure 3B). Interestingly, both hypo- and hypermorphic pancreas lengths were increased to 25% due to TCPMOH exposures.

Figure 2. Exocrine Pancreas Area.

Figure 2.

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Tg(ptf1a::GFP) Zebrafish embryos were imaged and measured at 96hpf following exposure to 50 nM TCPMOH, 50 nM TCPM, or 0.01% DMSO. Asterisks * designate statistically significant changes from controls (p<0.05). A) The area of fluorescent cells in the exocrine pancreas is presented in a bar graph as the Mean +/− Standard Error. An AVOVA with a Tukey (equal variances) post-hoc test was used to compare means. Mean exocrine pancreas Area of embryos exposed to 50 nM TCPMOH and 50 nM TCPM were reduced by 13% and 8.8%, respectively, compared to controls. B) Chi-square tests were used to compare incidence of hypomorphic and hypermorphic exocrine pancreas areas in each treatment group. Results are presented in a stacked bar graph, with the percent of areas that fell above the 90th percentile represented by gray bars (hypermorphic) and the percent of areas that fell below the 10th percentile represented by black bars (hypomorphic). n=26-32 embryos per exposure group.

Figure 3. Pancreas Length.

Figure 3.

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Tg(ptf1a:GFP) Zebrafish embryos were imaged and measured at 96hpf following exposure to 50 nM TCPMOH, 50 nM TCPM, or 0.01% DMSO. Asterisks (*) designate statistically significant changes from controls (p<0.05). A) The length of the pancreas is presented in a bar graph as the Mean +/− Standard Error. An AVOVA with a Games-Howell (unequal variances) post-hoc test was used to compare means. The mean pancreas length of embryos exposed to 50 nM TCPMOH was reduced by 6%, compared to controls. The mean pancreas length of embryos exposed to 50 nM TCPM was 2.7% larger than the controls. B) Chi-square tests were used to compare incidence of hypomorphic and hypermorphic pancreas lengths in each treatment group. Results are presented in a stacked bar graph, with the percent of lengths that fell above the 90th percentile represented by gray bars (hypermorphic) and the percent of lengths that fell below the 10th percentile represented by black bars (hypomorphic). Asterisks (*) designate statistically significant changes from controls (p<0.05). n=26-32 embryos per exposure group

3.3. Larval Morphology

Fish length and yolk sac area were measured at 4 dpf (Supplemental Figure 1). Fish length was quantified by measuring the rostral-to-caudal linear length of embryos. No significant difference in mean fish length was observed between treatment groups (p>0.05). Yolk sac area, an indicator of yolk nutrient utilization, was measured by tracing the outline of the yolk sac through the central (maximal) optical section. Similarly, to fish length, mean yolk sac area was not significantly impacted by exposure to TCPM and TCPMOH (p>0.05).

3.4. Gene Expression

RNA sequencing was performed to examine the genes most significantly impacted by developmental exposures to 50 nM TCPM or 50 nM TCPMOH from 24-100 hpf. The 10 most significantly upregulated and downregulated genes due to TCPM and TCPMOH exposures are shown in Supplemental Table 1. TCPMOH exposure significantly changed 547 genes (FDR <0.05), 215 of which were upregulated and 332 of which were downregulated. TCPM exposure significantly changed 419 genes (FDR<0.05), 137 of which were upregulated and 282 of which were downregulated (Figure 4A). The top 5 upregulated (Figure 4B) and top 5 downregulated (Figure 4C) genes between each exposure group are shown. Transcriptomic responses to developmental TCPM and TCPMOH exposures were strongly correlated (R2=0.903), and pathway analysis found downregulation of processes including retinol metabolism, circadian rhythm, and steroid biosynthesis (Figure 5). Processes related to genetic information processing, such as RNA degradation, DNA replication, and mismatch repair were upregulated, though nucleotide excision repair was downregulated. Pathways involved in metabolism of lipids, xenobiotics, and cofactors and vitamins were consistently downregulated by both TCPM and TCPMOH exposure. Other processes such as melanogenesis, toll-like receptor signaling, and circadian rhythm, were downregulated by exposures. All KEGG pathways significantly changed by exposure to TCPM and TCPMOH are displayed in Table 1, and statistics and full KEGG annotation are provided in Supplemental Table 2.

Figure 4. Gene Expression Summary.

Figure 4.

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A) Expression of 547 and 419 genes were significantly changed (p<.05) by TCPMOH and TCPM exposure, respectively, with 237 genes significantly changed by both. 101 genes were differentially expressed between TCPMOH and TCPM exposure and 35 genes were significantly altered across all group comparisons. B) The top 5 upregulated genes by treatment group. C) The top 5 down regulated genes by treatment group.

Figure 5. KEGG pathways changed by TCPM or TCPMOH exposure.

Figure 5.

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Transcriptomic responses to TCPM and TCPMOH are correlated (R2=0.903). Pathways in the upper right quadrant are upregulated by exposure to TCPM and TCPMOH. Pathways in the lower left quadrant are downregulated by exposure to TCPM and TCPMOH.

Table 1.

KEGG pathways significantly changed by TCPM and TCPMOH Exposures

Pathways Significantly Changed by TCPM Compared to Control
Upregulated Downregulated
Toll-like receptor signaling pathway Retinol metabolism
MAPK signaling pathway Circadian rhythm
RNA degradation Nucleotide excision repair
Metabolism of xenobiotics by cytochrome P450
Steroid hormone biosynthesis
Tyrosine metabolism
Metabolic pathways
Tryptophan metabolism
Oxidative phosphorylation
ABC transporters
Melanogenesis
Linoleic acid metabolism
Pathways Significantly Changed by TCPMOH Compared to Control
Upregulated Downregulated
MAPK signaling pathway Circadian rhythm
DNA replication Nucleotide excision repair
Cell cycle Retinol metabolism
Toll-like receptor signaling pathway ABC transporters
RNA degradation Tyrosine metabolism
Porphyrin and chlorophyll metabolism
Steroid biosynthesis
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To determine if observed morphological changes co-occurred with molecular changes, we examined expression of genes involved in pancreas development and function (Supplemental Table S3). Genes associated with the pancreatic hormone index, including glucagon (gcga), preproinsulin (insa), ghrelin (ghrl), and somatostatin 2 (sst2) were not significantly impacted by exposures to TCPM or TCPMOH. A key pancreas progenitor marker and transcription factor integral to pancreatic development, pancreatic and duodenal homeobox 1 (pdx1), was also not significantly changed. Expression of key exocrine pancreas digestive peptides, amylase, alpha 2 A (amy2a), carboxypeptidase A5 (cpa5), chymotrypsinogen b1 (ctrb1), trypsin (try), lipoprotein lipase (lpl), and elastases ela2l and ela3l, were not significantly changed by exposure to TCPM or TCPMOH.

Xenobiotic biotransformation processes are often key indicators of toxicity and mode of action for chemicals. The aryl hydrocarbon receptor (AHR) pathway is a major mediator of phase I detoxification through activation of cytochrome p450 enzymes (Khazaal et al., 2018). Pathway analysis revealed that “Metabolism of Xenobiotics by cytochrome P450” was significantly downregulated by TCPM exposure (p<0.001) but not TCPMOH exposure (p>0.05). To approach this at the gene-level, we examined genes involved in these highly inducible pathways to determine the metabolic and detoxification response to 50 nM TCPM and TCPMOH exposures (Supplemental Table S4). Cytochrome P450 enzymes including cyp1a, cyp1b1, byp1c1, cyp3a65, cyp2j20, and cyp2x8 were significantly downregulated, as was the aryl hydrocarbon receptor repressor (ahrra). The same pathway was found not to be significantly changed by TCPMOH exposure, though RNA sequencing showed several cytochrome P450 isoforms to be downregulated, including cyp2x8, cyp2y3, cyp2k18, and cyp3a65. No AHR pathway genes were significantly upregulated by TCPM or TCPMOH exposure (p>0.05).

The Nrf2 antioxidant response pathway mediates phase II detoxification by upregulating production of the endogenous antioxidant glutathione (GSH) (Hiebert & Werner, 2019). We examined genes involved in the Nrf2 antioxidant response pathway (Supplemental Table S5). Several glutathione-S-transferases (GSTs), gsto2 and gsta.1, were significantly downregulated by both TCPM (p<0.001 and p=0.003, respectively) and TCPMOH (p<0.001 and p=0.044, respectively) exposure. The GST omega 2 was the most significantly downregulated gene in the Nrf2 antioxidant response pathway for both TCPM and TCPMOH while the antioxidant enzyme prdx1 was significantly downregulated by only TCPMOH (p=0.001) exposure. The only genes involved in the Nrf2 pathway to be significantly upregulated by TCPM exposure were heat shock cognates, hsp70l and hsp70.2. Heat shock proteins, hsp90aa1.2 and hsp90aa1.1, and heat shock cognate, hsp70.3, were significantly decreased by TCPMOH exposure. Despite these expression changes to specific genes involved in the Nrf2 response, the overall “Glutathione Metabolism” KEGG pathway was not significantly changed by TCPM or TCPMOH exposure.

4. Discussion

The rapid rise in global prevalence of diabetes and other metabolic syndromes is a public health hazard and warrants investigation of potential causes of these diseases (National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, 2014). Recent evidence suggests chemical exposures during sensitive time periods may contribute to the developmental origins of diabetes by disrupting organogenesis of the developing pancreas (Inadera, 2013; Sant, Jacobs, et al., 2017; Simmons, 2006, 2007). Developmental exposures to TCPM and TCPMOH have been shown to induce embryonic mortality and deformities such as pericardial and yolk sac edema in zebrafish embryos (Navarrete et al., 2021; Yost et al., 2022). In the present study, we aimed to characterize the developmental toxicity of TCPM and TCPMOH to the zebrafish developing pancreas and gene expression. We hypothesized that embryonic exposures to TCPM and TCPMOH would disrupt pancreatic development and induce changes to gene expression. We found that exposure to 50 nM TCPMOH significantly reduced islet area and exocrine pancreas area in 4 dpf larvae. Exposure to 50 nM TCPM also resulted in a reduction in islet and exocrine pancreas area, though the reduction was not statistically significant.

4.1. Pancreatic Organogenesis is Sensitive to Toxic Exposures

Reduced beta cell mass and other pancreatic aberrant morphologies are often associated with diabetes or diabetic phenotypes, such as hyperglycemia, in humans and animal models (Bosco et al., 2010; Cabrera et al., 2006; Chen et al., 2014). β cells contained in the Islets of Langerhans are the only cells capable of secreting insulin and are thus critical to maintaining glucose homeostasis (Bosco et al., 2010; Sant, Jacobs, et al., 2017). While a direct mechanism of action between embryonic exposures to chemicals and the occurrence of diabetes later in life has not been confirmed, evidence suggests toxicants act to disrupt the sensitive developing pancreas via oxidative stress (Simmons, 2006). Developmental exposure to polychlorinated biphenyls, dioxins, phthalates, perfluorinated compounds, and various pharmaceuticals have been associated with diabetes in human epidemiological studies or associated with diabetic phenotypes, such as reduced beta-cell mass, in animal models(Brown et al., 2018; Chen et al., 2014; Jacobs et al., 2018; Sant et al., 2016; Sant et al., 2019; Timme-Laragy et al., 2015). Our study showed TCPMPOH is similarly capable of disrupting pancreatic organogenesis by reducing β cell mass and exocrine pancreas area.

4.2. Developmental Exposures to TCPM & TCPMOH Impact Xenobiotic Metabolism

Polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), DDT, and other organochlorine pesticides are known to induce oxidative stress and lipid peroxidation in organisms (Banerjee, 1999; Jennifer J. Schlezinger et al., 2006). Oxidative stress occurs when excessive generation of reactive oxygen species (ROS) overwhelm antioxidant defenses and may result in irregular gene expression, cell damage, cell death, and may contribute to disease development (Banerjee, 1999; Sant, Hansen, et al., 2017; Veith & Moorthy, 2018).

To assess potential detoxification mechanisms following TCPM and TCPMOH exposures, we examined pathways and genes involved in the AHR and Nrf2 antioxidant response pathways (Supplemental Tables S4 and S5). The AHR xenobiotic response pathway is a major Phase I biotransformation pathway, most commonly associated with induction of CYP1a enzymes. In our gene expression analysis, the KEGG pathway “Metabolism of Xenobiotics by Cytochrome P450” was significantly downregulated by TCPM exposure. TCPMOH exposure did not significantly downregulate the “Metabolism by Cytochrome P450 “pathway, though RNA sequencing did show downregulation of several cytochrome p450 isoforms. This indicates that TCPM may be a more competitive inhibitor of CYP1A enzymes than TCPMOH, and supports our recent findings (Yost et al., 2022). Exposure to some polychlorinated biphenyl (PCB) congeners, including 3,3 4,4 -tetrachlorobiphenyl (PCB 77) 3,3 ,4,4 ,5 -pentachlorobiphenyl (PCB 126) and 3,3 ,4,4 ,5,5 -hexachlorobiphenyl (PCB 169), have been found to uncouple the catalytic cycle of cytochrome p4501a activation, resulting in the release of reactive oxygen species and creating conditions of oxidative stress in fish (Jennifer J. Schlezinger et al., 2006). These non-ortho PCB congeners, inhibit and inactivate CYP1A enzymes in rats and fish by binding and uncoupling microsomal CYP1A (Besselink et al., 1998; Jennifer J Schlezinger et al., 2006; Schlezinger et al., 1999). The decrease in CYP1A activity displayed by TCPM may be due to the same inhibitory and inactivating effect displayed by these non-ortho PCB congeners.

Glutathione is the most abundant endogenous antioxidant in cells, though glutathione concentrations are naturally low in β cells. While the overall “Glutathione Metabolism” KEGG pathway was not significantly changed by exposure to TCPM or TCPMOH, several important genes involved in Phase II biotransformation and the Nrf2-mediated antioxidant response were induced. The Nrf2 transcription factor participates in cross talk with the AHR pathway and regulates the response to oxidative stress by binding to Antioxidant Response Elements (ARE) and governing the biosynthesis of GSH (Ohtsuji et al., 2008; Sant, Hansen, et al., 2017). Expression of glutathione-S-transferases, gsto2 and gsta.1 were significantly decreased by exposure to TCPM and TCPMOH, suggesting decreased glutathione utilization and conjugation. Ultimately, deficient expression of glutathione-S-transferases may decrease cellular ability to biotransform and remove TCPM, TCPMOH, and other xenobiotics.

4.3. Signaling Processes Significantly Disrupted by TCPM & TCPMOH Exposures

Development is a well-coordinated process governed by subtle and distinct cues mediating processes such as cellular division and proliferation, DNA replication, cell polarity, and differentiation (Hansen et al., 2020). When these processes are disrupted, adverse developmental outcomes such as structural and functional defects may occur. In our present study, the mitogen-activated protein kinase (MAPK) signaling pathway was the most upregulated KEGG pathway by exposure to TCPMOH and the second most upregulated pathway, behind Toll-like receptor signaling, for TCPM. The Cell Cycle KEGG pathway was also upregulated significantly by TCPMOH. Together, these cellular processes regulate processes of cell division and repair and suggest a cellular adaptive response to cytotoxic damage induced by TCPM and TCPMOH, which may contribute to disruption of development and gene expression (Martindale & Holbrook, 2002; Pucci et al., 2000).

Pathway analysis revealed processes related to Retinol metabolism were one of the most significantly downregulated responses to both TCPM and TCPMOH exposure (p<0.001). Retinol metabolism is an important process in developmental biology, governing outcomes such as reproduction, organogenesis, development, and apoptosis (Novák et al., 2008). Cytochrome p450 (Cyp1a), an important metabolic enzyme involved in Retinol processes, was downregulated significantly by TCPM, but not TCPMOH exposure. Expression of several retinoic acid metabolizing enzymes, including retinol dehydrogenases, rdh5 and rdh8a, and beta-carotene oxygenases, bco1 and bco1l, were decreased by exposure to TCPM and TCPMOH. These enzymes are needed to reduce All trans-retinaldehyde to the transcriptionally active retinoid species, all-trans-retinol and retinoic acid (Brun et al., 2016). Deficiency of these retinoid species and disruption of all-trans-retinoic acid signaling, in adult mice, displayed a 50% decrease in beta cell mass and enhanced beta cell apoptosis (Brun et al., 2015; Trasino et al., 2015). In Retinoic acid-deficient zebrafish embryos, development of both endocrine and exocrine pancreas cell types were decreased (Stafford & Prince, 2002). In their review of the role of retinoids in pancreatic islet and beta cell embryonic development, Brun et al, concluded that alterations of genes involved in retinol metabolism contributes to the development of embryonic defects, pancreatic dysmorphologies, and gluco-regulatory disfunction (Brun et al., 2016). In addition to the critical role RA and associated receptor signaling plays in embryonic pancreatic cell differentiation and proliferation (Brossaud et al., 2017), retinoids and retinol metabolism processes are needed to maintain beta cell mass and prevent beta cell apoptosis into adult life (Brun et al., 2016). In the present study, no genes related to pancreatic organogenesis were decreased by TCPM and TCPMOH, despite the observed morphological disruption of endocrine and exocrine pancreas development. This can likely be attributed to the decreased expression of retinol metabolism, and the important roles retinoid processes plays in promoting pancreatic cell differentiation and maintaining beta cell mass.

4.4. Metabolic Processes & Indicators of Endocrine Activity

In our KEGG pathway analysis, lipid metabolism processes related to steroid biosynthesis were downregulated by exposure to TCPM and TCPMOH. Also, Linoleic acid metabolism was down regulated by exposure to TCPM. Recent evidence has shown that disruption of circadian rhythm may influence lipid metabolism (Maradonna & Carnevali, 2018b). Additionally, several target genes in the peroxisome proliferator-activated receptor (PPAR) signaling pathway were significantly altered by exposure to TCPM and TCPMOH. PPAR signaling is important for xenobiotic and lipid metabolism and has shown to be disrupted by toxic exposures, namely perfluorinated compounds, phthalates, plasticizers, bisphenol A(BPA) related compounds, and fibrates (Maradonna & Carnevali, 2018a; Sant et al., 2019). Though expression of pparaa, ppardb, and pparg were not significantly changed, PPAR targets including fatty acid binding proteins (FABPs),fabp2 and fabp1a, were significantly down regulated by exposure to TCPM and TCPMOH. As lipid chaperones, FABPs regulate signaling and transport of steroids, including cholesterol (Furuhashi & Hotamisligil, 2008). These disruptions in steroid biosynthesis are closely associated with endocrine activity of chemicals, suggesting that TCPM and TCPMOH may have endocrine activity and potential for endocrine disruption similar to other organochlorine compounds (La Merrill et al., 2020). Further research exploring this potential using appropriate in vitro or sexually mature models should explore the endocrine activity of TCPM and TCPMOH.

Recent evidence has emerged implicating the disturbance of circadian rhythm as an influencer of lipid metabolism and PPAR signaling (Maradonna & Carnevali, 2018a; Sato et al., 2018). Circadian rhythm was the most significantly downregulated KEGG pathway for TCPMOH and the second most downregulated process for TCPM. The individual genes in the circadian rhythm pathway, Cry5 and Per2, were the two most downregulated genes for both xenobiotics. The expression of regulatory genes involved in processes such as glycolysis, lipid biosynthesis and metabolism, and oxidative phosphorylation are heavily influenced by circadian rhythm and disruption of circadian rhythm may result in metabolic disorders (Maradonna & Carnevali, 2018b; Sukumaran et al., 2010). Toxicants including Benzophenone 3, Tributyltin, Tetrabrominated biphenol A, and tris(1,3-dichloroisopropyl)phosphate have been shown to act on circadian rhythm and disrupt physiological processes related to metabolism and cell cycles (Zheng et al., 2021). In the present study, the disruption of circadian rhythm likely contributes to downregulation of other endocrine processes, including steroid biosynthesis and lipid metabolism, further implicating TCPM and TCPMOH as disruptors of endocrine activity and potential endocrine disruptors.

4.5. Implications, Future Directions, and Conclusions

The ubiquitous occurrence, potential for maternal transfer, high bioaccumulation, and high persistence of TCPM and TCPMOH in the environment are a public health concern. In this study, we used 50 nM concentrations of TCPM and TCPMOH for our waterborne exposures. These concentrations of TCPM and TCPMOH were selected because they were less than half of the NOAEL concentrations for survival and deformities in zebrafish embryos (Navarrete et al., 2021; Yost et al., 2022). It is important to note that transplacental transfer of TCPMOH (3.3-5.3%) and TCPM (0.55-1.2%) has been confirmed in whales (Kajiwara, Kamikawa, et al., 2008) and maternal transfer of TCPMOH has been observed during lactation in seals (Kajiwara, Watanabe, et al., 2008) and humans (Minh et al., 2001). Therefore, direct transplacental perfusion of the fetus and oral exposure in infants is likely. However, our waterborne exposures represent passive exposures at elevated concentrations, and therefore additional studies in a mammalian model are needed to characterize risk for humans.

In summary, developmental exposures to TCPM and TCPMOH have been shown to disrupt pancreatic organogenesis in zebrafish embryos by reducing beta cell mass and exocrine pancreas area. Changes to gene expression induced by TCPM and TCPMOH indicate a downregulation of genes involved in the xenobiotic response, steroid biosynthesis, and circadian rhythm, and increased expression of genes in pathways associated with cellular division and repair. Several key developmental pathways, including retinol metabolism, that direct pancreatic differentiation were altered and may contribute to the disruption of pancreatic organogenesis and function. Overall, exposure to TCPM and TCPMOH induced morphological and molecular effects at environmentally- and biologically-relevant concentrations that could contribute to the occurrence of metabolic diseases later in life in human and animal populations.

Supplementary Material

Supplemental Information

Acknowledgements:

Support for KES was provided by the National Institute of Environmental Health Sciences (K01 ES031640), and additional project support for this research was provided by San Diego State University’s University Grants Program (2021). This publication includes data generated at the UC San Diego IGM Genomics Center utilizing an Illumina NovaSeq 6000 that was purchased with funding from a National Institutes of Health SIG grant (#S10 OD026929). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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