1 Introduction
Marine pollution has become a public health concern owing to its potential impacts on marine life and humans. Many studies have focused on the toxicological effects of various chemical and physical stressors, such as pH, temperature, heavy metals, and endocrine-disrupting chemicals (
Donelson et al., 2010;
Dos Santos et al., 2020;
Delbès et al., 2022;
Taslima et al., 2022). However, the natural marine environment is polluted not only by wastewater treatment plant effluents (
Schwarzenbach et al., 2006;
Loos et al., 2009) but also by agricultural chemicals (
Moschet et al., 2014). Aquatic organisms are often exposed to complex mixtures of chemicals simultaneously (
Thrupp et al., 2018). Consequently, an increasing number of studies started focusing on the effects and mechanisms of these chemical mixtures.
According to a review published in 2001, seasonal hypoxic conditions have occurred along the coastal seabed during the past 15–20 years (
Diaz, 2001). Hypoxia is defined as a dissolved oxygen (DO) concentration of less than 2–3 mg O
2/L in marine environments (
Farrell and Richards, 2009). A review of the threshold levels of hypoxia for marine organisms, such as fish, crustaceans, grastropods, bivalves, polychaetes, cnidarians, and ctenophores, concluded that the mean LC
50 under hypoxia conditions for these species was 2.05 ± 0.09 mg O
2/L (
Vaquer-Sunyer and Duarte, 2008). Moreover, studies have suggested that hypoxia can impair multiple physiologic systems. Hypoxia exerts effects similar to those of endocrine disruptors (
Wu et al., 2003;
Lai et al., 2021;
Saha et al., 2022). It may cause embryonic developmental impairments in fish, such as increased embryonic mortality, altered hormone levels, and lowered hatching rates (
Wu et al., 2003;
Pollock et al., 2007;
Wu, 2009). Hypoxia has also been reported to alter fish swimming activities, including swimming speed (
Domenici et al., 2013).
Endocrine-disrupting chemicals (EDCs) are a diverse group of synthetic and natural compounds that are widespread in aquatic environments. 17α-ethinylestradiol (EE2), an EDCs, has been reported to impact embryonic development in fish, reducing embryonic survival rates, increasing teratogenesis rates, and delaying hatching (
Brown et al., 2007;
Hu et al., 2017;
Qin et al., 2023). Furthermore, a study showed that EE2 uptake in killifish (
Fundulus heteroclitus) is exacerbated under hypoxic conditions (
Blewett et al., 2013), suggesting that the interactive effects of EE2 and hypoxia on organisms may be more severe than those of a single factor. However, the interaction effects of EE2 and hypoxia on embryonic development are poorly understood. Based on the aforementioned findings: (1) hypoxia may exert endocrine disrupting effects in organisms, and (2) the accumulation of EE2 increases under hypoxic condition. Therefore, it is hypothesized that combined exposure to the endocrine disruptor EE2 in hypoxic condition may cause interactive effects (including antagonistic, additive, or synergistic) that differ significantly from the sum of their individual effects on fish embryonic development. These effects may influence various aspects of embryonic development, including developmental patterns, heart rate, hatching, and larval locomotion.
In this study, two concentrations of EE2, 71 ± 5 and 668 ± 35 ng/L, were applied for exposure. Numerous studies have reported an unbalanced global distribution of EE2 concentrations. In freshwater samples from rivers, EE2 concentrations ranged from below detection limits to 33.5 ng/L in Austria, China, Hungary, Slovenia, and the USA, whereas Argentina, Brazil and Span reported much higher concentrations of more than 100 ng/L (
Brossa et al., 2005;
Klaic and Jirsa, 2022). For water samples near the estuary, EE2 concentrations ranged from below detection limits to 97.7 ng/L in the Douro River Estuary, Portugal (
Ribeiro et al., 2009). In marine environments, EE2 concentrations have been reported from below the detection limit to less than 50 ng/L, with higher values of 75 ng/L reported in Italy (
Pojana et al., 2004;
Almeida et al., 2020). Therefore, concentrations below 100 ng/L are considered environmentally realistic for toxicological experiments (
Lee et al., 2014). By contrast, the threshold of hypoxic conditions ranges from 2 to 5 mg/L depending on the organisms (
Vaquer-Sunyer and Duarte, 2008;
Gobler and Baumann, 2016). For marine fish, specifically, DO levels from 1.6 to 2.5 mg/L at pH 7.5 were considered hypoxic to assess early-life sensitivities (
DePasquale et al., 2015). Therefore, 71 ng/L EE2 was used as an environmentally realistic concentration, 668 ng/L EE2 served as a positive control, and a DO level of 1.8 mg/L was used as the hypoxia condition.
In the present study, we used marine medaka (
Oryzias melastigma) as an animal model to address the embryonic developmental toxicity induced by the interaction between EE2 and hypoxic condition. Marine medaka is a fish species native to Asia. The transparency of its embryos facilitates the observation of morphological changes during embryogenesis, making it an ideal model for studying the biological impacts of various pollutants (
Kim et al., 2016;
Horie and Takahashi, 2021). Detailed differences in embryogenesis were observed and recorded, as the duration of marine medaka embryogenesis is approximately 9–13 d (
Iwamatsu, 2004;
Wang et al., 2020), compared to the shorter duration of approximately 3 d in zebrafish embryogenesis, another common fish model (
Kimmel et al., 1995). This longer embryogenesis duration allows researchers to more easily record and compare morphological changes in marine medaka. Several endpoints were used to evaluate potential impairments during embryogenesis. First, developmental pattern was assessed. Second, heart rate was measured to determine developmental delays. Third, the hatching process including hatching time and hatching rate was evaluated to assess developmental delays and teratogenicity induced by combined exposure of EE2 and hypoxia. Finally, larval locomotion was measured to identify the lasting harmful effects on embryonic development. Our study indicated that combined exposure to EE2 under hypoxic conditions induced various effects compared to EE2 or hypoxia alone. Notably, an antagonistic effect was observed for hatching time under combined exposure to low EE2 concentrations in hypoxia, whereas an additive effect was observed for eye pigmentation development under combined exposure to high EE2 concentrations in hypoxic conditions.
2 Materials and methods
2.1 Chemicals and equipment used
HPLC grade dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA) and stored at 4 °C. EE2 (purity ≥ 98%; CAS No. 57-63-6) was purchased from Sigma Chemical Company (St. Louis, MO, USA). The EE2 stock solution was dissolved in DMSO, and the working EE2 solutions were prepared in 1 L glass bottles with nominal concentrations of 125 and 1000 ng/L by diluting the stock solution with Milli-Q water. The DMSO control solution was prepared using the same procedure but without the addition of EE2. A Leica S6 E Stereo Zoom Microscope and Nikon SMZ1270 were used, along with the imaging software NIS-Elements. A Heal Force Tri-Gas Incubator (HF100) was purchased from Heal Force International Trading Co., Ltd. (Shanghai, China) to maintain hypoxic conditions. An RNA extraction kit (TransZol Up Plus RNA Kit, CAS No. ER501) was purchased from TransGen Biotech (Beijing, China). PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect Real Time) (CAS No. RR047A) and TB Green® Premix Ex TaqTM (Tli RNaseH Plus) (CAS No. RR420A) were purchased from Takara Bio Inc. (Kyoto, Japan) for cDNA synthesis and PCR amplification.
2.2 Fish embryo maintenance and experimental setup
Marine medaka embryos were obtained from the City University of Hong Kong. Embryos were collected from the bottom of the tank within 1 h of morning light activation using a fine mesh net. They were then washed under running tap water by gentle kneading along the outer surface of the net before being transferred to Petri dishes. Embryos were cleaned under a microscope. Unfertilized and unviable embryos were discarded, and only fertilized, healthy embryos no older than 8 h post-fertilization (hpf) were used for exposure. In this study, an environmentally relevant concentration (71 ng/L) and a high EE2 concentration (668 ng/L) were used for exposure. A Heal Force Tri-Gas Incubator (HF100) was set to 95% N2 and 5% CO2 in a hypoxic environment. After exposure, the DO levels of the exposure solutions were detected by a DO meter, and were determined to be 1.8 mg/L. The embryos were exposed to six conditions until hatching: (1) control (0.004% of DMSO), (2) low concentration of EE2 (71 ng/L in 0.004% of DMSO, EE2_L), (3) high concentration of EE2 (668 ng/L in 0.004% of DMSO, EE2_H), (4) hypoxia group (1.8 mg/L DOfor the first 2 d of the experiment), (5) low concentration EE2 + hypoxia (EE2_L + hypoxia), and (6) high concentration EE2 + hypoxia (EE2_H + hypoxia). Each experiment included four replicates with 100 embryos per replicate. After 48 h of hypoxia exposure, all hypoxic groups were removed from the chamber. The embryos were reared under the following husbandry conditions: pH 7.4, 26 ± 1 °C, 7.2 ± 0.2 mg/L O2, 25‰ salinity, and a 14-h light/10-h dark cycle. The exposure solution for each group was renewed daily. All treatments were maintained until hatching. Larvae were reared in cubic tanks (n = 4, dimensions: 15 cm × 15 cm × 15 cm) for locomotion measurements at 3 d post-hatching (dph).
2.3 Measurement of EE2 exposure concentration
Water samples were collected from Petri dishes at 1, 3, 5, and 7 d of exposure and analyzed by liquid chromatography-tandem mass spectrometry. Water samples were subjected to solid-phase extraction using Oasis® WAX cartridges (500 mg, 6 mL). Methanol (5 mL × 2) and Milli-Q water (5 mL × 2) were sequentially added to precondition the cartridge. After loading the water samples into each cartridge (flow rate: one drop per second), the cartridge was washed with 10 mL of Milli-Q water, dried under vacuum for 30 min, and eluted with 10 mL of methanol. A gentle stream of high-purity nitrogen was used to concentrate the eluate to 20 µL at 40 °C. Replicate procedural blanks (Milli-Q water) and procedural recoveries (Milli-Q water spiked with 50 ng of EE2) were included in each batch of sample analyses. The average procedural recovery of EE2 was 76% ± 0.07%.
An Agilent 1290 Infinity ultra-performance liquid chromatograph (Agilent, Palo Alto, CA, USA) interfaced with a 5500 QTRAP® mass spectrometer (AB Sciex, Foster City, CA, USA) using multiple reaction monitoring in negative ion electrospray mode was used to determine EE2 concentrations. The Agilent Eclipse Plus C18 guard column (2.1 mm i.d. × 5 mm L., 1.8 µm) and the Agilent Eclipse Plus C18 analytical column (2.1 mm i.d. × 50 mm L., 1.8 µm) were employed. Briefly, a 10 µL aliquot of the extract was injected into the analytical column. The mobile phase consisted of 0.1% NH4OH in Milli-Q water (solvent A) and acetonitrile (solvent B), with a flow rate of 0.3 mL/min. The gradient program was as follows: 50% solvent B stable for 1 min, increased to 90% over 3 min, maintainedfor 4 min, and returned to 50% within 0.1 min. During instrumental analysis, a quality control sample (10 ng/mL EE2 standard solution) was injected every 12 samples to monitor instrument stability. The standard deviation of the quality control was 0.55 ng/mL, indicating good instrument stability.
2.4 Determination of embryonic development
The observation period spanned from 0 d post-fertilization (dpf) to 7 dpf, with data collection during the entire embryogenesis period, defined as the time from fertilization to hatching (
Iwamatsu, 2004;
Kinoshita et al., 2009). This period was divided into three stages for comparing embryonic development: (1) pre-eye pigmentation period (0 dpf to the day when less than half of the embryos displayed eye pigmentation), (2) eye pigmentation period (from the day more than 50% of embryos displayed eye pigmentation to the day no embryos hatched), and (3) hatching period (from the day embryos began hatching to the day hatching ceased). The embryos were observed daily in the morning using a Leica S6 E Stereo Zoom Microscope. The developmental periods of the control were used as standards for comparison.
2.5 Measurement of heart rate, hatching delay, and hatching rate
The heart rate was measured at 3 dpf and on the first day of hatching. For each treatment replicate, a 40-s video was recorded using a Nikon SMZ1270 with the imaging software NIS-Elements. Ten embryos were randomly selected from each treatment replicate, and their heart rates were recorded for 30 s. The recorded data were multiplied by two to express the results as beats per minute.The hatching delay and hatching rate of the embryos were recorded from 0 to 20 dpf. Hatching delay was recorded, and the percentage of hatched embryos relative to the total number of embryos was calculated as the final hatching success.
2.6 Larval locomotion assessment
Twenty-four 3-d-old larvae were randomly selected from each treatment group for larval locomotion assessment. Larval locomotion data were captured using the Noldus behavior recording system equipped with Ethovision software version 3.1 (Noldus Information Technology, Leesburg, VA, USA) and analyzed using R software (version 4.2). The active area of the larvae was set as the area of the bottom of the wells in 24-well plates, and the coordinates of origin point 0 were adjusted to the center of the well. Onelarva and 1 mL of clean artificial seawater were placed in each well. The program for larval locomotion was set as follows: (1) acclimation: 10 min, 100% intensity level of light, no data collection, (2) lasting stimulation: 15 min, 100% intensity level of light, with data collection, (3) transitional stimulation, 10 min, 5 min in dark switching to 5 min of 100% intensity level of light, with data collection. These three periods of behavioral data were analyzed and presented as the means of speed.
2.7 Total RNA extraction, cDNA cloning and RT-qPCR
Thirty 5-d-old embryos from each replicate were randomly collected and pooled in RNAlater
TM Stabilization Solution (Thermo Fisher Scientific) for storage at −80 °C until total RNA extraction (
n = 4, total 4 × 6 × 30 = 720 embryos). Total RNA was extracted using the TransZol Up Plus RNA Kit (TransGen Biotech), following the manufacturer’s instructions. Briefly, frozen samples were thoroughly ground into a powder under in liquid nitrogen. Subsequently, 1 mL of TransZol Up and 0.2 mL RNA Extraction Agent were added for homogenization. After a 5-min incubation at room temperature and a 15-min centrifugation at 10000 × g, the upper transparent aqueous phase was collected. This phase was mixed with an equal volume of absolute ethanol, washed with the kit washing solution, and dissolved in RNase-free water for subsequent analysis. The total RNA concentration was quantified using a spectrophotometer (Thermo Scientific NanoDrop
TM 2000 Spectro-photometers). RNA with an A260/A280 ratio of no less than 1.8 was used for cDNA synthesis. Subsequently, 1 μg of RNA was used for cDNA synthesis using PrimeScript™ RT Reagent Kit (Takara Bio Inc) and gDNA Eraser (Perfect Real Time), following the product manual. Quantitative polymerase chain reaction (qPCR) was performed with TB Green® Premix Ex Taq
TM (Tli RNaseH Plus) (Takara Bio Inc) using a C1000 Touch Thermal Cycler under CFX96
TM Real-Time PCR Detection System (Bio-Rad Laboratories Inc., Germany). The cycling conditions were as follows: (1) denaturation: 95 °C for 30 s, (2) annealing and extension: 95 °C for 5 s and 60 °C for 30 s, for a total of 40 cycles.
O. melastigma 18S rRNA served as the reference gene for normalization (
Kim et al., 2013). The threshold cycle (
Ct) value was determined using CFX96
TM Real-Time System CFX Maestro
TM software version 2.3 (Bio-Rad), and ∆
Ct values were calculated as ∆
Ct =
Ct (reference gene) –
Ct (target gene). The fold change in relative gene expression was determined using the 2
–∆∆Ct method (
Livak and Schmittgen, 2021).
2.8 Statistical analysis
All data were presented as the mean ± SEM. Data were shown as bar plots generated using GraphPad Prism 8 software (GraphPad, USA). Different letters indicated a significant differences among multiple comparisons performed using a one-way ANOVA followed by Duncan’s test (p < 0.05).
3 Results
3.1 EE2 and hypoxia exposure altered embryonic development
The nominal low- and high-dose EE2 concentrations of 125 and 1000 ng/L, the actual measured concentrations were 71 ± 5 and 668 ± 35 ng/L, respectively. The DO in the exposure solution was 1.8 mg/L. Eye pigmentation started at 2 dpf in the control group (Fig.1), whereas both EE2_L and EE2_H delayed this onset by 1 d (starting at 3 dpf). Moreover, exposure to hypoxia and EE2_L + hypoxia resulted in a 2-d (starting at 4 dpf), whereas EE2_H + hypoxia caused a significant 3-d delay in eye pigmentation (starting at 5 dpf).
To further determine the alterations in embryogenesis caused by EE2 and hypoxia exposure, embryonic development was assessed at three stages. Our results showed that exposure to either hypoxia or EE2 prolonged the pre-eye pigmentation period compared with that in the control group (Fig.1). This aligns with the observed delay in onset of eye pigmentation (Fig.1). During the eye pigmentation period, only the high dose of EE2 caused a significant extension compared to the controls. There were no significant changes observed during the hatching period. Collectively, these results suggest that EE2 and hypoxia mainly affect the pre-eye pigmentation period of embryogenesis.
3.2 EE2 and hypoxia exposure led to a reduction in embryonic heart rate
Our results showed that at 3 dpf, EE2, hypoxia, and their combination significantly decreased the heart rate of embryos (to an unobservable heartbeat) compared with that in the control groups (Fig.2). However, on the day of the first fish hatching, the EE2_L and EE2_L + hypoxia groups exhibited a significant reduction in the heart rate (8.71% and 11.87% with p = 0.0170 and p = 0.0026, respectively), whereas the hypoxia groups showed no significant change (p = 0.1614) compared to the control group (Fig. 2B). Furthermore, comparing the high EE2 concentration with the control group, EE2_H group showed no significant difference (p = 0.0823), but the EE2_H + hypoxia group showed a significant 11.35% reduction (p = 0.0033).
3.3 The hypoxic and EE2 exposures reduced the hatching rate of embryos
Our results showed that hypoxic exposure significantly delayed the initiation of hatching by 2 d (Fig.3). EE2 exposure induced a dose-dependent hatching delay, and combined EE2 and hypoxia exposure induced a 1 d delay compared to EE2 exposure alone (Fig.3). Moreover, our results showed the EE2 exposure caused a reduction in the hatching rate at high concentrations. The combined exposure of a high concentration of EE2 and hypoxia significantly increased the hatching rate (83.26% ± 5.72%) compared with that of the EE2_H group (60.10% ± 5.55%), but no significant changes were observed compared to the control group (Fig.3).
3.4 Effects of EE2 and hypoxia exposure on larval locomotion
Three larval speed periods were recorded for assessment. In the first 15 min of the light stable period, only the high-dose EE2 group showed a significant 33.85% decrease in larval speed compared with that in the control groups (Fig.4). The hypoxic and EE2-H + hypoxia groups showed a decreasing trend but this was not statistically significant. No significant changes were observed during the second and third periods (Fig.4 and 4C). These results suggest that the reduction in larval locomotion primarily result from EE2 exposure.
3.5 EE2 or hypoxia exposure altered the gene expression related to embryonic development, heart development, nervous function and behavior
Based on the observed phenotypic changes, we summarized the alterations in embryonic development, heart rate, and larval locomotion caused by EE2 and hypoxic exposure. qPCR analysis was performed to understand the molecular changes underlying these adverse effects. Genes related to heart development (Ephrin B2 [EFNB2]), synaptic function and behavior (leucine-rich repeat transmembrane neuronal 1 [LRRTM1] and Syntaxin binding protein 1 [STXBP1B]), and eye development and function (Nuclear receptor subfamily 2 group E member 3 [NR2E3], Syntaxin 3 [STX3], and Thioredoxin-related transmembrane protein 3 [TMX3]) were also included. Our results showed that EE2 exposure had no effect on the gene expression of EFNB2, but hypoxia exposure significantly inhibited its expression, with fold changes of 0.295 ± 0.211, 0.285 ± 0.271, and 0.196 ± 0.088 in the hypoxia, EE2_L + hypoxia, and EE2_H + hypoxia groups, repectively (Fig.5). For synaptic function and behavior, both EE2 and hypoxia exposure suppressed the expression of LRRTM1 and STXBP1B with fold changes below 0.5 (Fig.5). A similar result was observed in eye development and function-related genes; EE2 and hypoxia exposure reduced the expression of NR2E3, STX3, and TMX3, with fold changes below 0.6 (Fig.5).
3.6 Effects induced by the combined exposure of EE2 and hypoxia
The effects of the combined exposure of EE2 and hypoxia were further compared to the control, hypoxia, and EE2 exposure groups. a: indicates a significant difference between exposure groups (hypoxia, EE2_L, EE2_L + hypoxia, EE2_H, and EE2_H + hypoxia) and control groups; b: indicates a significant difference between exposure groups (EE2_L, EE2_L + hypoxia, EE2_H, and EE2_H + hypoxia) and hypoxia groups; c: indicates a significant difference between the combined exposure and EE2 exposure (EE2_L vs. EE2_L + hypoxia, EE2_H vs. EE2_H + hypoxia) (Tab.1 and Tab.2). Our results showed that the EE2_H + hypoxia group presented a significant delay in the appearance of eye pigmentation compared with the other three groups (control, hypoxia, and EE2_H). Additionally, the EE2_L + hypoxia group showed a significant delay in hatching time compared with the other three groups (control, hypoxia, and EE2_L) (Tab.1). The additive, synergistic, and antagonistic effects are defined as follows, additive: the combined effect is equal to the sum of the individual effects of each pollutant; synergistic: the combined effect is greater than the sum of the individual effects of each pollutant, and antagonistic: the combined effect is less than the sum of the individual effects of each pollutant (
Jin et al., 2019;
Mainka and Magdalena, 2022;
Sures and Nachev, 2022). The delayed times of hypoxia, EE2_H, and EE2_H + hypoxia groups in the appearance of eye pigmentation were 2, 1, and 3 d (= 1 + 2), respectively. The delayed times in hatching were 1.5, 1.5, and 2.5 d (< 1.5 + 1.5). These results indicate an additive effect induced by the combined exposure of EE2 and hypoxia at high concentrations in eye pigmentation development and an antagonistic effect induced by the combined exposure of EE2 and hypoxia at low concentrations during hatching.
4 Discussion
4.1 Combined exposure of EE2 and hypoxia induced an antagonistic effect on the hatching time and affected the hatching process
Although increasing attention is being paid to the combined harmful effects of pollutants, few studies have focused on the combination of EE2 and hypoxia. A study using male Fathead minnows (
Pimephales promelas), exposed to a mixture of EE2, 17β-estradiol (E2), 4-tert-nonylphenol, 4-tert-octylphenol, and bisphenol A (BPA) under hypoxia (DO level: 1.44 to 1.75 mg/L) for 14 d, showed no significant change in the level of vitellogenin (
Brian et al., 2009). However, our results showed that newly fertilized embryos exposed to EE2, hypoxia, and combination of EE2 and hypoxia exhibited a significant delay in embryonic development, especially in the appearance of eye pigmentation. The difference in results may be because of (a) different fish species (
Li et al., 2015;
Spurgeon et al., 2020), fathead minnows are freshwater species, whereas marine medaka are seawater species used in our study; (b) different exposure scenarios (
Dourson et al., 2004;
Wang et al., 2021), Brian’s study used sexually mature fish, but newly fertilized embryos were used in our study, which are more sensitive during exposures; (c) different concentrations used (
Dornberger et al., 2016;
Connell et al., 2024), highest concentration of EE2 used in Brain’s study was 1.68 ng/L, which is lower than the concentrations used in our study (71 ± 5 and 668 ± 35 ng/L of EE2).
A study that examined zebrafish embryos exposed to a combination of EDCs and hypoxia revealed similar changes in embryonic development. In this study, zebrafish embryos were subjected to combined exposure to BPA and hypoxia (2.0 ± 0.5 mg/L O
2) from 1 hpf to the late hatching period. The results indicated significant decreases incardiac parameters, including heart rate and red blood cell velocity (
Kubo et al., 2004;
Cypher et al., 2018). Additionally, marine medaka embryos exposed to perfluorobutane sulfonate (PFBS) and hypoxia (DO level: 1.7 mg/L) from fertilization to 15 dpf exhibited significant reductions in survival, heart rate, and hatching rate (
Tang et al., 2020). Although the EDCs studied differed from those in our study, both BPA and perfluorobutane sulfonate have been reported to possess estrogenic properties in various organisms (
Seachrist et al., 2016;
Li et al., 2020), suggesting that combined exposure to estrogenic EDCs and hypoxia may elicit similar developmental effects.
4.2 Combined exposure of EE2 and hypoxia leads to an interactive effect in embryonic development compared with that of EE2 or hypoxia exposure alone
Considering the complexity of the natural environment, researchers have highlighted the necessity of chemical mixture exposure studies rather than focusing on the effects of single chemicals (
Carlin et al., 2013). In particular, studies of chemical mixture exposure must address several important questions to understand their effects, including: (a) the impacts of individual chemicals, (b) interacting effects of chemical mixture, and (c) cumulative exposure effects of the chemical mixture (
Braun et al., 2016). In this study, we evaluated the effects of individual factors and factor mixtures to understand their interaction.
In a typical chemical mixture exposure experiment, it is important to determine how the chemicals interact and whether their effects are additive, antagonistic, or synergistic (
Lazarevic et al., 2019). An
in vivo study on pejerrey fish (
Odontesthes bonariensis) demonstrated that the larval survival rate showed a declining trend at 4 dph under single E2 or EE2 exposure, and combined exposure to E2 and EE2 showed a significant decline in larval survival rate, indivative of an additive effect (
Gárriz et al., 2015). Our study showed that the combined exposure to EE2 and hypoxia induced additive effect on eye development (eye pigmentation) and antagonistic effects on hatching time. These results suggest that the effects induced by the combined exposure to EE2 and hypoxia cannot be simply defined as additive, antagonistic, or synergistic astheir impact varies in different biosystems. Previous studies have reported different EE2 accumulation patterns in fish organs. For example, EE2 accumulates in the gall bladder, followed by the carcass, gut, liver, gill, and spleen (
Blewett et al., 2013). Another study showed that hypoxic conditions can significantly change the tissue-specific accumulation of EE2 in fish (
Blewett et al., 2013). Moreover, a study showed that hypoxia inhibited the expression of the estrogen receptor (ER) in ER-positive cell lines of breast cancer (
Wolff et al., 2017). Additionally, the 20-h estradiol and hypoxia exposure has been reported to synergistically degrade the expression of ERα in MCF-7 human breast cancer cells (
Yi et al., 2009). Although these results were obtained from breast cancer cells, they suggest that hypoxia can interact with estrogen receptors and may further affect organisms. Hypoxic exposure may alter the accumulation and distribution patterns of EE2 in fish and could potentially affect the expression of estrogen receptors. This interaction may alter estrogenic activity of EE2 in specific organs, leading to differential gene expression and adverse effects in fish. However, the gene expression of ERα in marine medaka embryos may not be detected during early embryogenesis. One study detected its expression only as early as 6 dpf (
Chakraborty et al., 2011). Therefore, the function of estrogen receptors in the early embryonic stage under hypoxic exposure remains poorly understood This hypothesis requires further evidence, particularly through embryonic exposure experiments, to enhance our understanding in future studies.
4.3 Exposure to EE2 and hypoxia altered the gene expression related to embryonic development and behavior
Based on the phenotypic results, we found that combined exposure to EE2 and hypoxia induced an additive effect on eye development during embryogenesis. Therefore, we hypothesized that this exposure might also alter gene expression related to embryonic development, eye development, and eye function. We conducted a qPCR analysis of genes associated with these observed phenotypes.
EFNB2 belongs to ephrin protein family and is highly affected by hypoxic conditions (
Himanen et al., 2007). This proteins family is essential for neurous system function and angiogenesis (
Brantley-Sieders and Chen, 2004;
Butler and Tear, 2007;
Reber et al., 2007).
EFNB2 is primarily expressed in arteries (
Gerety et al., 1999), and
EFNB2 knockout mice have defects in arteries and veins at the capillary linkage between the head and yolk sac during angiogenesis (
Wang et al., 1998). This finding supports our result that exposure to EE2 and hypoxia reduced heart rate during embryogenesis.
For genes related to eye function and behavior,
LRRTMA1 is highly expressed in the adult human brain and is involved in the stimulation of excitatory synaptogenesis (
Linhoff et al., 2009;
Winther and Walmod, 2014). More importantly, targeted deletion of
LRRTM1 results in a chronic reduction in excitatory synaptic function and may further affect cognitive, social, and sensorimotor gating deficits reminiscent of schizophrenia (
Karimi et al., 2021).
STXBP1B is a homolog of
STXBP1 in zebrafish (
Suo et al., 2021). A study on
STXBP1 haploinsufficient mice showed a reduction in cortical inhibitory neurotransmission and dysfunctional in cognitive, psychiatric, and motor functions (
Chen et al., 2020). Furthermore, homozygous
STXBP1B mutants in zebrafish caused neurodevelopmental abnormalities and decreased locomotor activity under a “dark-flash” visual stimulus system (
Grone et al., 2016;
Liu et al., 2021). These findings suggest that
LRRTM1 and
STXBP1B may play a role in the reduced larval locomotion observed under EE2 or hypoxic exposure.
In addition, we investigated eye development and functional genes.
NR2E3 is a photoreceptor-specific orphan receptor involved in rod differentiation and is related to rod photoreceptor-specific genes (
Cheng et al., 2004). An
NR2E3 knockout mouse study showed a defect in retinal morphology and abnormalities within the outer nuclear layer of the retina (
Webber et al., 2008).
STX3 is also associated with eye functions, such as the fusion of vesicles in the photoreceptor plasma membrane, transportation of rhodopsin, and release of synaptic vesicles (
Zulliger et al., 2015). The deletion of
STX3 in retinal cells results in a rapid degeneration and a mis-trafficking of photoreceptor outer segment proteins (
Kakakhel et al., 2020). Another study using
STX3 knockout mice indicated that
STX3 is necessary for retinal photoreceptor cell survival (
Janecke et al., 2021).
TMX3 was reported to play a role in delaying eye growth (
Guerra and Molinari, 2020). This gene is expressed in the retinal neuroepithelium and lens epithelium, and TMX haploinsufficiency can affect eye morphology (
Chao et al., 2010). The reduction in these genes aligns with our findings that exposure to EE2 and hypoxia led to a delay in eye pigmentation. Impairments in larval locomotion may also be attributed to dysfunction of the eye structure. However, it should be noted that there was no synergistic alteration in these genes under the combined exposure to EE2 and hypoxia, suggesting that more complex molecular mechanisms may be involved in the combined effects.
5 Conclusions
Exposure to EE2 or hypoxia impaires embryonic development, resulting in delayed the developmental processes, reduced heart rates, decreased hatching rates, and suppressed larval locomotion. Further gene expression analysis highlighted the involvement of genes such as EFNB2, LRRTM1, STXBP1B, NR2E3, STX3, and TMX3 in the observed phenotypic changes. This gene cluster could be added to the gene list for evaluating the effects of EE2 or hypoxia.
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