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Published in final edited form as: Chemosphere. 2025 Mar 16;376:144300. doi: 10.1016/j.chemosphere.2025.144300

Tert-butylphenol exposure alters cartilage and bone development in zebrafish

Haley Jo Brashears 1, Kayla Lea 1, Syed Rubaiyat Ferdous 1, Subham Dasgupta 1, Eric H Baldwin 1, Lisa J Bain 1,*
PMCID: PMC13445940  NIHMSID: NIHMS2197678  PMID: 40096755

Abstract

Phenolic antioxidants, such as 2,4-di-tert-butylphenol (2,4-DTBP), 2-tert-butyl phenol (2-BP), and 4-tert-butyl phenol (4-BP), are additives used in domestic water pipes, food packaging, paints, and other industrial products. As additives, they can leach from products and are frequently found in both environmental and human biological samples. Previous studies have demonstrated that 2,4-DTBP exposure can impair the differentiation of human iPS cells into somite- and sclerotome-like cells, and reduce key processes involved in osteoblast formation. Therefore, the goal of this study is to determine if 2-BP, 4-BP, 2,4-DTBP, and its metabolite 3,5-di-tert-butylcatechol (3,5-DTBC) impacts the development of cartilage and bone in vivo, using zebrafish as a model organism. Zebrafish embryos were exposed to increasing concentrations of each of the four chemicals from 1 hours post fertilization (hpf) until 5 days post fertilization (dpf), and analyzed for markers of bone and cartilage development. At their highest concentrations tested, both 2-BP and 2,4-DTBP altered axial skeleton formation, with 76% and 61% of the zebrafish showing spinal curvatures, respectively. To corroborate these changes, the expression of marker transcripts were examined. 2-BP exposure reduced mRNA expression of the bone mineralization marker sparc by 1.6-fold. In contrast, 2,4-DTBP increased sparc transcript expression by 1.4-fold. All four compounds significantly upregulated sox9a, a chondrogenesis marker, between 1.4- to 5-fold. Changes in tail cartilage formation were noted using Alician blue staining, with 2,4-DTBP reducing width, length, and cartilage area of the tail, while 2-BP reduced the tail width but with increased the tail base, yielding a more straightened tail. Principle component analysis (PCA) demonstrated associations between sox9a, sparc, nrf2a, reactive oxygen species (ROS), and tail cartilage measurements, particularly in the 2,4-DTBP exposures, suggesting the involvement of nrf2a signaling in impairing cartilage formation. Overall, the study shows that each of the phenolic antioxidants differentially affects the development of bone and cartilage structures in zebrafish.

Keywords: 2,4-di-tert-butylphenol; Nrf2; zebrafish; cartilage; 2-tert-butylphenol; 4-tert-butylphenol

Graphical Abstract

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1. Introduction

Phenolic antioxidants are used as additives to products to increase shelf life by reducing oxidative reactions (Ji et al., 2023; R. Liu & Mabury, 2020). In particular, the phenolic antioxidants 2,4-di-tert-butylphenol (2,4-DTBP), 2-tert-butylphenol (2-BP), and 4-tert-butylphenol (4-BP) are heavily used, but information on their toxicity is scarce. For example, 2,4-DTBP is used in car seats, food packaging plastics, and several personal care products (R. Liu & Mabury, 2019a; Qian, 2018; Wu & Venier, 2023). One of its most common uses is in domestic drinking water pipes. 2,4-DTBP has been shown to leach from plastic high density polyethylene (HDPE) and cross-linked polyethylene (PEX) pipes into drinking water, at concentrations ranging from 0.02 – 0.76 μM, depending on the pipe age and frequency of use (Z. H. Liu et al., 2017; Lund et al., 2011; Skjevrak et al., 2003). Further, 2,4-DTBP can migrate from processing and packaging materials into baby cereal and formula, with one study finding median levels of 0.14 μM and 0.08 μM 2,4-DTBP, respectively, in these products (Du et al., 2023). 2-BP is used during fragrance and pesticide production, while 4-BP is used in food lubricants, paints, resins, and coatings (Yadav & Doshi, 2002). Indeed 4-BP has been detected in surface waters in Germany and rivers in Japan at concentrations up to 0.4 μM (Inoue et al., 2002; Janousek et al., 2020).

Due to their high use and detection frequency in the environment, both 2,4-DTBP and 4-BP have been detected in human samples. For example, a study in U.S. found 100% detection frequency in 60 random urine samples, with the highest level detected at 69.9 ng/mL (0.34 μM). This same study also found that a metabolite of 2,4-DTBP, 3,5-di-tert-butylcatechol (3,5-DTBC), was found in 27% of samples (R. Liu & Mabury, 2019b). Additionally, a study of 36 pregnant women demonstrated that 2,4-DTBP was detected in 100% of the maternal plasma, 78% of cord plasma, and 86% of placenta samples (Du et al., 2019). 4-BP has also been detected in urine with the highest concentration being 6.7 ng/mL (0.046 μM) (Kuklenyik et al., 2003). Therefore, it has become essential for us to understand whether these SPAs elicit toxicity and impair developmental processes.

In vivo, there is little known about the toxicity of the butylated phenols. Newborn and juvenile rats exposed for 28 days to 300mg/kg/day of 2-BP had decreased locomotor activity (Hirata-Koizumi et al., 2005). Zebrafish embryos exposed to 2,4-DTBP up to 28 hours post fertilization (hpf) had decreased locomotor activity and increased anxiety-like behavior (W. Liu et al., 2022). Zebrafish exposed to 4.8 μM 2,4-DTBP had decreased heart rates and an overall increase in malformation rate up to 96 hpf (Shi et al., 2022). Similarly, exposure to 4-BP at concentrations ranging from 3–12 μM increased skeletal defects and reduced body length in exposed zebrafish (Wang et al., 2023). In vitro, human induced pluripotent stem cells exposed to 2,4-DTBP have decreased differentiation into somite-like cells and sclerotome-like cells, which can be differentiated into chondrocytes and osteoblasts (Dương et al., 2023). While chondrocyte formation was not examined in this study, the impaired differentiation due to 2,4-DTBP exposed was corroborated by reductions in the osteogenic transcripts OSX and RUNX2, and lowered Alizarin Red staining, an indicator of mineralization (Dương et al., 2023). Collectively, these studies suggest that the tert-butylphenol antioxidants and are associated with impaired cellular differentiation and altered skeletal structure. Therefore, it is critical that we understand how these chemicals alter bone and cartilage formation.

Zebrafish are an ideal vertebrate model for investigating effects of chemicals on early developmental processes, due to their external fertilization and rapid development (Kimmel et al., 1995). Somite formation starts at 11 hpf, and is concluded by approximately 24 hpf (Holley & Nüsslein-Volhard, 2000). Zebrafish form bone by both intramembranous ossification, and by endochondral ossification, in which bone replaces a cartilage template (Weigele & Franz-Odendaal, 2016; Witten et al., 2017). The first cartilaginous structures are formed in the jaw of zebrafish at 2 days post fertilization (dpf), fully formed cartilage in the trunk and tail is seen at 5 dpf, and skeletal bone structures are seen at 12 dpf (Bird & Mabee, 2003).

The goal of this study was to determine if the phenolic antioxidants 2-BP, 4-BP, and 2,4-DTBP, as well as its metabolite 3,5-DTBC, impair development in zebrafish larvae. Zebrafish embryos were exposed to increasing concentrations of 2-BP, 4-BP, 2,4-DTBP, or 3,5-DTBC until 5 dpf. Results indicate that exposure to 2,4-DTBP and 2-BP cause changes in skeletal morphology, and alter the expression of transcripts related to chondrocyte formation and bone mineralization. Changes in reactive oxygen species and nrf2 levels suggest a mechanism of action for the butylphenols.

2. Methods

2.1. Chemicals

2,4-DTBP was purchased from TCI (Portland, Oregon; purity >97%), and 2-BP (purity >99%), 4-BP (purity >99%), and 3,5-DTBC (purity >99%) from ThermoFisher Scientific. All chemicals were dissolved in DMSO in washed amber glass bottles, at stock concentrations of 200 μM. Stocks were stored at −20°C.

2.2. Zebrafish husbandry and embryo procurement

Dr. Robyn Tanguay’s lab (Oregon State University) kindly provided adult zebrafish (Tropical 5D) that were raised in the Aquatic Animal Research Laboratory (AARL) at Clemson University. All procedures were conducted under an approved IACUC protocol. The zebrafish were kept on a flow through system under standard laboratory conditions (28°C with 14 h light: 10 h dark photo cycle). For each experiment, three mating pairs were placed in mating tanks overnight. Eggs from each tank were collected, mixed together, and cleaned within 15 minutes of fertilization.

2.3. Zebrafish chemical exposures

Individual eggs were placed in cell culture treated 96 well plates. Embryos at the 2-cell stage were exposed to 2,4-DTBP (0, 1, 5, or 10 μM), 2-BP (0, 5, 10, or 20 μM), 4-BP (0, 5, 10, or 20 μM), or 3,5-DTBC (0, 0.1, 1, or 2 μM) until 5 dpf. At 24 hpf, water was renewed with the inclusion of 0.003% phenylthiourea (ThermoFisher) to prevent pigment formation. Zebrafish embryos were observed every 24 hours, and mortality and hatching recorded. Exposures with 4-BP and 3,5-DTBC was renewed every 24 hours. Exposures were repeated on at least three separate days, and each concentration had 4 – 5 replicates (each replicate is a 96-well plate). From each microplate, approximately 10 fish were used for Alcian blue and Alizarin red staining, another 15 – 20 fish were used for RNA extraction, 15 – 20 fish were used to investigate reactive oxygen species (ROS) levels, and the remainder used for morphological assessment.

2.4. Assessment of developmental toxicity

At 5 dpf, a stereomicroscope was used to record malformations for all embryos, including spinal defects, swim bladder inflation, and yolk and pericardial edema. Zebrafish were then euthanized with 1 g/L buffered tricaine methane sulfonate (MS-222). Lateral view images from 8–10 random larvae per chemical per concentration were taken on a Leica M165FC microscope. Fifteen larvae were placed in microcentrifuge tube with TRIreagent (Sigma Aldrich) and stored in −80°C for RNA extraction.

2.5. Cartilage and bone staining with Alician blue and Alizarin red

Zebrafish were stained using a two-color acid-free cartilage and bone stain based on published protocols (Walker & Kimmel, 2007). Euthanized fish were fixed in 2% paraformaldehyde for 1 hr followed by a 100 mM Tris/10 mM MgCl2 wash. The cartilage stain, 0.04% Alcian Blue/120 mM MgCl2 was added overnight. After rehydration with EtOH, a 3% H2O2/0.1% KOH bleaching solution was added. Fish were washed in 25% glycerol/0.1% KOH and the incubated in 0.01% Alizarin stain for 2 hrs. Finally, fish were incubated with 50% glycerol/0.1% KOH to destain, and stored fresh 50% glycerol/0.1% KOH at room temperature prior to imaging.

For imaging cartilage in the tail, stained 5 dpf fish (eight random fish per concentration per chemical) were placed on their lateral side and photographed. ImageJ was used to measure tail base width, width of the tail at its widest point, distance (length) between the tip of the trunk to end of the tail, overall area of the tail, and the integrated density value (IDV) of tail cartilage levels. For imaging craniofacial bones and cartilage, stained fish were placed in depression molds on their ventral side. ImageJ was used to measure Meckel’s, palatoquadrate, and ceratohyal cartilage lengths, the distance between Meckel’s and ceratohyal cartilage, the angles between cartilage structures, and areas of anterior and posterior otoliths

2.6. Quantitative PCR

Transcript levels of bone mineralization (sparc/osn), cartilage formation (sox9a), skeletal muscle formation (myod), and oxidative stress (nrf2a, gstp, cat) were assessed using qPCR. RNA extraction was done on 15 – 20 zebrafish per exposure group, and concentration and purity were determined using a NanoDrop (Thermo Scientific). MMLV reverse transcriptase (Promega, Madison, Wisconsin, USA) were used to synthesize cDNA. To assesses gene expression, real time PCR was performed on the Bio-Rad CFX96 (Hercules, CA, USA) using SYBR green (Applied Biosystems, Foster City, CA, USA) and gene specific primers. For each transcript, a standard was made by PCR using composite cDNA from all experimental groups, as verified by gel electrophoresis as a single band of the correct size. The standard was then diluted into five concentrations from 10−3 - 10−7 ng/μL to determine primer efficiency, and a melt curve was obtained from each run to determine non-specific primer binding. The housekeeping genes actin and gapdh were used to normalize expression. Fold expression relative to controls was assessed using the delta-delta Ct method. Primer sequences and conditions are in Supp. Table 1.

2.7. DCF to assess ROS levels

DCFH-DA (2’,7’-dichlorofluorescein diacetate) binds to intracellular ROS, upon which the dye is oxidized to 2’7’-dichlorofluorescein (DCF). At 5 dpf, exposed zebrafish (n = 10–14 per concentration per exposure group) are incubated in 10 μM DCFA-DA for 30 minutes, washed to remove excess dye, anesthetized in MS-222, and DCF levels assessed on a Leica M165FC fluorescent microscope.

2.8. Statistical analyses

Hatching, mortality, morphological changes, and DCF levels were averaged by chemical and exposure group. For analysis of qPCR data, the geometric mean of gapdh and actin were used as the reference gene, and the ΔΔCt method was used to quantify gene expression changes (n = 4 – 5 pools of zebrafish per exposure group). ImageJ was used to measure length, distance, angles and areas of craniofacial bone and cartilage structures (n = 7– 8 images per exposure group). All data were analyzed in GraphPad Prism 10 using ANOVA followed by Tukey’s (p < 0.05). Results are depicted as the mean and standard deviation for each group. Principle coordinate analysis (PCA) for each exposure group was conducted in GraphPad Prism 10 and the resultant biplot and correlations matrices are presented.

3. Results

3.1. Hatching and morphological changes in exposed zebrafish

Zebrafish were exposed for 5 days to increasing concentrations of 2-BP, 4-BP, 2,4-DTBP, and 3,5-DTBC and observed every 24 hours to assess survival and hatching. There were no reductions in viability at any concentrations tested for 2,4-DTBP (0, 1, 5, and 10 μM), 3,5-DTBC (0, 0.1, 1, and 2 μM), 2-BP (0, 5, 10, and 20 μM), and 4-BP (0, 5, 10, and 20 μM) (Fig. 1A). However, there was a significant delay in hatching rate at the highest concentration of 2,4-DTBP (Fig. 1B). While the fish were still alive, there were unable to break through the chorion. Exposure to the metabolite 3,5-DTBC did not alter hatching (Fig. 1C). Concentrations of 20 μM 2-BP (Fig. 1D) and 20 μM 4-BP (Fig. 1E) caused an initial delay in hatching, but by the end of the study, these fish hatched at the same frequencies as the controls.

Fig. 1. Viability and hatching rates after exposure to 2,4-DTBP, 3,5-DTBC, 2-BP, and 4-BP.

Fig. 1.

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Zebrafish embryos were exposed for five days to 2,4-DTBP (0, 1, 5, and 10 μM), 3,5-DTBC (0,0.1, 1, and 2 μM), 2-BP (0, 5, 10, and 20 μM), or 4-BP (0, 5, 10 and 20 μM) from 1 hpf to 5 dpf. Mortality and hatching rates were monitored every 24 hours. Data is presented as the average ± SD from a 96-well microplate (n = 3 microplates per concentration per exposure group). Statistical differences (*; p < 0.05) were assessed by ANOVA followed by Tukey’s.

Zebrafish were also individually examined for yolk edema, pericardial edema, and spinal deformities (Fig. 2A). There was a significant increase in pericardial edema due to 2,4-DTBP exposure and an increase following 2-BP exposure (Fig. 2B). Yolk sac edema was elevated in all four exposure groups at their highest concentrations of 10 μM 2,4-DTBP, 2 μM 3,5-DTBC, 20 μM 2-BP, and 20 μM 4-BP (Fig. 2C). Frequencies of spinal deformities, including spinal curvatures and tail kinks, were increased in both the 10 μM 2,4-DTBP and 20 μM 2-BP exposure groups, such that 61% and 72% of the fish displayed these malformations, respectively (Fig. 2D).

Fig. 2. Increased incidence of malformations in zebrafish embryos.

Fig. 2.

Fig. 2.

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Zebrafish embryos were examined for morphological changes at 5 dpf. Representative images are presented following exposure to 2,4-DTBP (0, 1, 5, and 10 μM), 3,5-DTBC (0, 0.1, 1, and 2 μM), 2-BP (0, 5, 10, and 20 μM), or 4-BP (0, 5, 10 and 20 μM) (A). Frequencies of pericardial edema (B), yolk sac edema (C), and spinal deformities (D) were determined. Data is presented as the average ± SD from a 96-well microplate (n = 3 microplates per concentration per exposure group). Statistical differences (*; p < 0.05) were assessed by ANOVA followed by Tukey’s.

3.2. Alterations in cartilage development

Since the incidence of spinal deformities was high in the 2-BP and 2,4-DTBP exposure groups, and because 2,4-DTBP has been previously shown to inhibit sclerotome formation, osteoblast differentiation and calcium deposition (Dương et al., 2023), we asked whether cartilage formation and/or indicators of bone mineralization would also be inhibited in vivo. In zebrafish, the first cartilaginous structures are formed in the jaw at 2dpf, while hypertrophic chondrocytes and fully formed cartilage in the trunk and tail are seen at 5 dpf. It is not until 12 dpf in which most of the skeletal bone structure can be seen (Hammond & Schulte-Merker, 2009; Schilling & Kimmel, 1997).

Therefore, the 5 dpf exposed zebrafish were stained with Alizarin Red and Alcian Blue, and cartilage structures in the trunk and tail were assessed including the width of the tail (W), the tail base width (B), distance between trunk and tip of the tail (T-T), and overall cartilage intensity in the tail (area), as measured by integrated density value (IDV). The images show that 2,4-DTBP appears to reduce the width and area of the tail in a dose-responsive manner (Fig. 3). Similarly, exposure to 2-BP also appears to alter the overall tail size (Fig. 3). To measure and quantitate cartilaginous structures in the tail, ImageJ was used. Exposure to 5 and 10 μM 2,4-DTBP (medium and high concentrations), or 10 and 20 μM 2-BP (medium and high concentrations) reduced the overall width of the tail up to 1.3-fold (Fig. 4A). With the highest 20 μM concentration of 2-BP, the width at the base of the tail was significantly increased, yielding a more straightened tail (Fig. 4B). Likewise in both 2,4-DTBP and 2-BP, the distance between the tip of the trunk to the end of the tail (T-T) was also reduced by up to 1.4-fold (Fig. 4C). Interestingly, T-T distance was also reduced in 4-BP exposed fish at all concentrations (Fig, 4C), but the overall tail shape was preserved. As above, the IDV values as a measure of cartilage staining in the tail was reduced in the 5 and 20 μM 2,4-DTBP exposure groups by 1.2- and 1.6-fold, respectively (Fig. 4D), while 2-BP exposure at 10 and 20 μM reduced IDV values by 1.3- and 1.6-fold, respectively (Fig. 4D).

Fig. 3. Images of stained trunk and tail cartilage.

Fig. 3.

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Zebrafish were exposed to 2,4-DTBP (1 – 10 μM), 3,5-DTBC (0.1 – 2 μM), 2-BP (5 – 20 μM), or 4-BP (5 – 20 μM) for 5 days and then co-stained with Alcian blue and Alizarin red. Representative images of trunk and tail cartilage are presented, indicating tail width (W; orange), the width of the tail base (B; white), distance between trunk and tip of the tail (T-T; green), and overall tail area (area; black dash).

Fig. 4. Cartilage formation in the tails is dysregulated due to tert-butylphenol exposure.

Fig. 4.

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Zebrafish was stained with Alcian blue to examine changes in body and tail cartilage structure, length, and area. Width of the tail (A), tail base width (B), trunk to tail distance (C), and IDV, an overall assesment of tail cartilage intensity (D) were measured using the line and polynomial functions in ImageJ. Distances are expressed in μm. Average distance or IDV (n = 8 images per exposure group) and standard deviation are shown. Statistical significance (*) was determined using two way ANOVA followed by Tukey’s (p < 0.05).

To confirm changes in cartilage following 2,4-DTBP exposure, several cartilage structures in the lower jaw, along with anterior and posterior otoliths, were measured (Supp. Fig. 1). The highest concentration of 10 μM 2,4-DTBP reduced the length of Meckel’s (M), palatoquadrate (PQ), and ceratohyal cartilage (CH), (Supp. Fig.2A–C) along with the M-CH distance (Supplementary Fig. 2). In contrast, 4-BP increased the values of both the angle between M – PQ and between CH – CH cartilage (Supp. Fig. 2E–F), which are indicative of a wider head. The Alizarin red stained otoliths were used to assess changes in mineralization. In the high 10 μM 2,4-DTBP exposure group, both anterior and posterior otolith area was reduced by 1.2-fold (Supp. Fig. 3A–B). In contrast, anterior and posterior otolith area in the 4-BP exposure group had a dose-dependent increase in size by 1.2-, 1.3-, and 1.4-fold in the 5, 10, and 20 μM exposure groups, respectively (Supp. Fig. 3A–B).

3.3. Changes in chondrogenic, osteogenic, and myogenic transcript levels

The changes in spinal curvature, cartilage formation in the caudal fin and craniofacial area, and otoliths suggest that the development of cartilage and/or calcium mineralization was being altered due to phenolic antioxidant exposure. Therefore, mRNA levels of sox9a (chondrocyte differentiation marker), myod1 (marker of skeletal myogenesis), sparc (or osn; marker of bone mineralization), and nrf2a (marker of oxidative stress) were assessed in 5 dpf zebrafish using qPCR. The results show that 2,4-DTBP increases levels of sox9a, myod, sparc, and nrf2a transcripts by 1.3- to 1.8-fold in the 10 μM exposure group (Fig. 5A). Similarly, 3,5-DTBC increased sox9a and myod mRNA levels by 1.8- to 2.2-fold, but instead reduced sparc mRNA levels by 3.3-.fold (Fig. 5B). While sox9a transcripts were also increased in both the 2-BP and 4-BP exposure groups (Fig. 5C, 5D), neither one of these chemicals altered expression of other transcripts.

Fig. 5. Altered expression of chondrogenic, osteogenic, and myogenic genes in 5 dpf zebrafish.

Fig. 5.

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Zebrafish were exposed to 2,4-DTBP (1 – 10 μM), 3,5-DTBC (0.1 – 2 μM), 2-BP (5 – 20 μM), or 4-BP (5 – 20 μM) for five days. Transcript levels of sox9a (chondrogenesis marker), sparc (bone mineralization marker), myod1 (skeletal muscle marker) and nrf2a (reactive oxygen species transcription factor) were determined with qPCR. Data was normalized using geometric mean of the reference genes actin and gapdh. Fold changes were calculated using the 2^ddCt method and expressed as the mean for each group. Statistical significance (*) was determined using ANOVA followed by Tukey’s (p < 0.05). 15 – 20 zebrafish were collected for one replicate; each chemical and concentration has 4 – 5 replicates.

3.4. Reactive oxygen species (ROS) levels are increased after 2,4-DTBP or 3,5-DTBC exposure

2,4-DTBP, 2-BP, and 4-BP are used as antioxidants in inert commercial products. To investigate whether their antioxidant functions would also occur in a living organism, 5 dpf zebrafish were incubated in DCFH-DA (2’,7’-dichlorofluorescein diacetate), which is oxidized to 2’7’-dichlorofluorescein (DCF) after interacting with intracellular ROS, and produces a green fluorescent color (Fig. 6A; Supp. Fig. 1). Levels of DCF are increased by 2.3- and 1.5-fold in the highest concentrations of both 2,4-DTBP and 3,5-DTBC, respectively (Fig. 6B). Interestingly, exposure to 2-BP reduced DCF levels by 1.6-fold (Fig. 6B).

Fig. 6. 2,4-DTBP and 3,5-DTBC increase DCF levels in zebrafish.

Fig. 6.

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After five days of exposure, zebrafish were incubated in DCFA-DA. Representative images of 2,4-DTBP exposed fish are shown (A). Representative images for 3,5-DTBC, 2-BP, and 4-BP fish can be found in Supp. Fig. 4. The fluorescence from the resultant DCF was measured in whole fish, minus their yolk sac (B). Data is presented as the average relative fluorescent units (RFU), setting the mean control value at an RFU of 1 (n = 10 fish per concentration per exposure group). Statistical significance (*) between control and treatment groups was determined using ANOVA followed by Tukey’s (p < 0.05).

3.5. Correlations between chemical-induced changes in cartilage and ROS

To investigate relationships between chemical exposure, ROS, and cartilage development, principle component analysis (PCA) was used. The model contained data for cartilage (sox9a mRNA expression, tail cartilage distances and collagen IDVs), mineralization (sparc mRNA expression), and reactive oxygen species (nrf2a, gstp, and cat levels, DCF expression). The biplot for 2,4-DTBP shows that the 10 μM exposure fish are grouped together in the far right side of the graph (green circle; Fig. 7), which correlates with expression of ROS, nrf2a, its target genes gstp and cat, and sparc (Fig. 7). This grouping is driven along the PC1 axis, which accounts for 51.5% of the variance. The 5 μM 2,4-DTBP exposure group clustered together (red circle) in the bottom of the biplot, driven more by PC2, which accounts for 14.5% of the variance (Fig. 7). With PC1 and PC2, the cumulative proportion of variance is 66%. The control (black circle) and 1 μM 2,4-DTBP (orange circle) groups are clustered together in the left side of the graph, driven by Sox9a expression and tail cartilage measurements (Fig. 7).

Fig. 7. Principle component analysis (PCA) biplot for 2,4-DTBP.

Fig. 7.

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A biplot was constructed from PCA using data from cartilage (sox9a mRNA expression, cartilage distances and tail IDV), bone mineralization (sparc mRNA expression), and reactive oxygen species (nrf2a. gstp, cat mRNA levels, DCF expression) measurements for each 2,4-DTBP exposure group (n = 8 fish). Paired samples were used when possible. Control fish are designated by beige dots, 1 μM 2,4-DTBP fish by orange dots, 5 μM 2,4-DTBP fish by red dots, and 10 μM 2,4-DTBP fish by green dots. A black, orange, red, and green circle surround the location of the control, 1, 5, and 10 μM 2,4-DTBP fish, respectively.

For 2-BP, the patterns are quite different. The10 μM and 20 μM 2-BP exposed zebrafish (red and green circles, respectively) also group together towards the right side of the biplot (Fig. 8). These clusters are driven by sox9a, gstp, catalase, and sparc along PC1, representing 46.6% of the variance. The control (black circle) and 5 μM 2-BP (orange circle) exposed fish cluster together on the left side of the biplot, driven by ROS, nrf2a, and tail cartilage measurements (Fig. 8). Correlation matrices for all four chemicals also confirm associations between ROS, nrf2a, sparc, and M-PQ angle for both 2,4-DTBP and 4-BP (Supp. Table 2).

Fig. 8. Principle component analysis (PCA) biplot for 2-BP.

Fig. 8.

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A biplot was constructed from PCA using data from cartilage (sox9a mRNA expression, cartilage distances and tail IDV), bone mineralization (sparc mRNA expression), and reactive oxygen species (nrf2a. gstp, cat mRNA levels, DCF expression) measurements for each 2-BP exposure group (n = 8 fish). Paired samples were used when possible. Control fish are designated by beige dots, 5 μM 2-BP fish by orange dots, 10 μM 2-BP fish by red dots, and 20 μM 2-BP fish by green dots. A black, orange, red, and green circle surround the location of the control, 5, 10, and 20 μM 2-BP fish, respectively.

4. Discussion

The tert-butylphenols are used as synthetic antioxidants in products such as drinking water pipes, plastics, paints, fragrances, and other industrial products (R. Liu & Mabury, 2020; Qian, 2018; Yadav & Doshi, 2002). One member of this class, 2,4-di-tert-butylphenol (2,4-DTBP), has been shown to inhibit somite and sclerotome formation in vitro (Dương et al., 2023), which are precursor structures to cartilage and bone. The results of the present study suggest that in developing zebrafish, exposure to 2,4-DTBP and 2-tert-butylphenol (2-BP) alters cartilage formation potentially due to dysregulated Nrf2 signaling. While 4-tert-butylphenol (4-BP) exposure also reduces cartilage-based structures, its mode of action appears not to be dependent on Nrf2.

Exposure to 2,4-DTBP and 2-BP dysregulates cartilage formation

Zebrafish were exposed for five days to one of the three tert-butylphenols or to 3,5-di-tert-butylcatechol, a known metabolite of 2,4-DTBP (R. Liu & Mabury, 2019b), starting at the two-cell stage. Both 2,4-DTBP and 2-BP exposure resulted in a dose-dependent increase in spinal deformities, which included lordosis, kyphosis, or scoliosis. The incidence of deformities was at 61% in the highest concentration of 2,4-DTBP and 72% in the highest concentration of 2-BP, both of which did not yield an increase in death. In other studies, zebrafish exposed to 4.8 μM 2,4-DTBP up to 4 dpf had an overall increase in malformation rate (Shi et al., 2022). Similarly, exposure to 4-BP at concentrations ranging from 3–12 μM increased skeletal defects and reduced body length in exposed zebrafish (Wang et al., 2023). The exposures in the current study were all performed in tissue-culture treated 96-well plates, while exposures in other studies were conducted in glass beakers. The concentrations of 2,4-DTBP and 4-BP that resulted in significant skeletal deformities are slightly different between the studies, likely due to increased binding of tert-butylphenols to the tissue culture plates.

In zebrafish and many other vertebrates, the axial skeleton, including cartilage and bone, is derived from somites and the sclerotome (Morin-Kensicki et al., 2002). During development, somite formation begins at 10.5 hours post-fertilization, the first cartilaginous structures are seen in the head and jaw at 2 dpf, and by 5 day post-fertilization, cartilage in the axial skeleton and caudal fin or tail is developed (Bird & Mabee, 2003; Schilling & Kimmel, 1997). The most posterior three caudal vertebrae are modified to form the base of the tail or caudal fin (Bird & Mabee, 2003), from which the initial cartilaginous tail fin processes radiate out. Thus, examining structures in the tail allows for an overall assessment of chemical effects on cartilage formation. The data shows that zebrafish exposed to increasing concentrations of 2,4-DTBP have reductions in several cartilage structures in the tail. These include significantly reduced tail or caudal fin width, reduced distance between the trunk and the tail to assess caudal fin length, and reductions in the overall intensity of cartilage staining in the tail, as indicated by IDV values. Similarly, fish exposed to 2-BP have reductions in tail width, trunk-to-tail distance and IDV values. However, the base of the tail is actually wider in the 2-BP exposure groups, suggesting that 2,4-DTBP and 2-BP have similar, but not equivalent effects on cartilage formation.

Marker transcripts associated with cartilage and bone formation were also examined. Sox9a is a transcription factor involved in chondrocyte differentiation (Y. L. Yan et al., 2002), while sparc is needed for calcium mineralization (Maurer et al., 1995; Termine et al., 1981). 2-BP exposure reduced mRNA expression of sparc by 1.6-fold, while 2,4-DTBP increased sparc transcript expression by 1.4-fold. At the highest concentration of 2,4-DTBP, sox9a expression increased slightly, by 1.4-fold. Sox9a levels were also increased in 2-BP and 3,5-DTBC exposures, by 5- and 2.2-fold, respectively. Studies using other chemicals have also shown correlations between changes in sox9a expression and spinal curvatures, and alterations in sparc expression in conjunction with increased spinal curvatures, slower bone formation, and decreased bone mineralization (Li et al., 2023; R. Yan et al., 2023). Using principle component analysis (PCA), the generated biplots demonstrate dose-dependent differences due to 2,4-DTBP and 2-BP exposure. Both the 5 μM and 10 μM 2,4-DTBP exposure groups are distinctly separate from the control group. These changes are driven by altered sparc expression, ROS production, and nrf2 signaling. Similarly, both the 10 μM and 20 μM 2-BP exposure groups are separate from the controls, driven by sparc and sox9 expression. Thus, the gene expression results corroborate the cartilage formation data, and suggest that zebrafish exposed to 2,4-DTBP or 2-BP have dysreglated cartilage development.

Nuclear factor erythroid 2-related factor 2 (nrf2) is the key transcription factor in the antioxidant response. During oxidative stress, nrf2 phosphorylation and nuclear translocation induces expression of genes that reduce oxidative stress, including catalase (cat) and glutathione S-transferase (gstp) (Ray et al., 2012). Nrf2 signaling also regulates stem cell differentiation. In human embryonic stem cells (hESCs), NRF2 activity is high, but is reduced during their differentiation. Thus, NRF2 activation delays hESC differentiation and maintains stemness, while NRF2 inhibition enhances differentiation (Jang et al., 2014).

The data indicates that nrf2 expression is increased in the highest 2,4-DTBP exposure group. Similarly, DCF expression as a measure of ROS levels is also increased in the 2,4-DTBP exposure group. These results suggest that 2,4-DTBP might delay the differentiation of stem cells into cells important for cartilage formation. Beyond playing a role in stem cell differentiation, nrf2 appears to regulate bone and cartilage formation. For example, cells with hyperactivated Nrf2 showed impaired differentiation of osteoblasts (Yoshida et al., 2018). In concordance, MC3T3-E1 preosteoblast cells that have Nrf2 knocked down demonstrate enhanced osteoblast differentiation, mineralization, and expression of osteoblast marker genes (Park et al., 2014). Overall, it appears that increased Nrf2 expression results in reduced osteoblast differentiation and mineralization. Indeed, human studies correlate de novo mutations that cause NRF2 induction with delayed bone age and shorter stature (Huppke et al., 2017). Changes in Nrf2 expression and activity also has effects on cartilage formation. For example, when Nrf2−/− mice are treated to develop osteoarthritis, they have much greater cartilage loss than wild type mice (Cai et al., 2015). Similarly, Nrf2 loss increases cartilage destruction in mice that have rheumatoid arthritis (Maicas et al., 2011). These results might suggest that changes in differentiation seen in the 2,4-DTBP-exposed zebrafish are, in part, due to the activation of nrf2 signaling.

PCA demonstrated associations between bone and cartilage marker genes with ROS levels and nrf2 expression, suggesting a mechanism of action. However, each of the tested phenolic antioxidants differentially affects differentiation. The 4-BP exposures caused no significant malformations, but did reduce cartilage expression in the tails, and reduce the trunk to tail distance. While 2-BP exposure resulted in significant spinal deformities, reduced cartilage expression in the tails, but increased the width of the tail base, yielding a more straightened tail. Although the reasons for these differences are unknown, one possibility could be due to differences in metabolism and elimination. Glucuronidation is a major biotransformation pathway for phenols, but the position of the tert-butyl group on 2-BP could cause steric hindrance, thereby increasing the half-life of 2-BP (Laakia et al., 2009; Williams et al., 2003). This would also be expected to be the case for 2,4-DTBP. Furthermore, several studies have found that 4-BP is converted into a catechol, but there is no evidence that would suggest 2-BP behaves similarly (Nishimaki-Mogami et al., 2022).

These findings indicate that cartilage formation and bone mineralization are dysregulated by 2,4-DTBP and 2-BP exposure. At present, these chemicals cannot be ruled out as potential public health and environmental concerns.

Supplementary Material

1

Highlights.

  • 2,4-DTBP and 2-BP caused skeletal malformations in zebrafish

  • Four different tert-butylphenols upregulated sox9a expression

  • 2,4-DTBP reduces the amount of width, length, and area of the tail

  • Dysregulated nrf2a signaling is involved in impairing cartilage development

Acknowledgements:

We thank John Smink and Scott Horman from Clemson’s Aquatic Animal Research Laboratory for assistance with zebrafish husbandry.

Funding:

Funding was provided by the National Institutes of Health (P20 GM121342)

Footnotes

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CRediT authorship contribution statement

Haley Jo Brashears: Methodology, Validation, Formal analysis, Investigation, Data curation, Writing – original draft, Writing – review & editing, Visualization. Kayla Lea: Methodology, Validation, Formal analysis, Investigation, Data curation. Syed Rubaiyat Ferdous: Methodology, Validation, Formal analysis, Investigation, Data curation. Subham Dasgupta: Conceptualization, Supervision, Formal analysis, Writing – review & editing. Eric H. Baldwin: Methodology, Validation, Formal analysis, Investigation, Data curation. Lisa J. Bain: Conceptualization, Supervision, Validation, Formal analysis, Writing – review & editing, Visualization, Funding acquisition.

Declaration of Competing Interest The authors declare that they have no known competing financial and/or personal interests.

Declaration of interests

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Lisa Bain reports financial support was provided by Clemson University. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Data availability

Data will be made available on request.

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Associated Data

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Supplementary Materials

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NIHMS2197678-supplement-1.docx (337.6KB, docx)

Data Availability Statement

Data will be made available on request.

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