Abstract

Background

   Tris (2-chloroethyl) phosphate (TCEP), a widely used flame retardant,
   is widespread in the environment and potentially harmful to organisms.
   However, the specific mechanisms of TCEP-induced neurological and
   reproductive toxicity in fish are largely unknown. Turbot (Scophthalmus
   maximus) is cultivated on a large scale, and the emergence of
   pollutants with endocrine disrupting effects seriously affects its
   economic benefits. This study aimed to investigate the toxic effects of
   TCEP on turbot by integrating physio-biochemical and transcriptomic
   analyses.

Results

   TCEP exposure induced severe neuroendocrine disrupting effects in
   turbot. Firstly, the hormone levels of luteinizing hormone (LH),
   follicle-stimulating hormone (FSH), estradiol (E2), and
   11-ketotestosterone (11-KT) were significantly decreased under
   prolonged TCEP stress, which may have a negative impact on normal
   reproductive function. We identified and summarized representative
   differentially expressed genes (DEGs) and their functions, such as
   endocrine system and oxidative stress. Pathway enrichment showed that
   the toxicological characteristics of TCEP on turbot were neuroendocrine
   regulation disorders, including oxidative phosphorylation, apoptosis,
   steroid biosynthesis, GnRH signaling pathway and so on. Weighted gene
   co-expression network analysis (WGCNA) also revealed key genes involved
   in these pathways. Among these genes, those encoding the components of
   the electron transport chain presented an initial increase in
   expression followed by a decrease, indicating that TCEP stress might
   affect mitochondrial function and lead to cell damage. This finding was
   also supported by the upregulation of apoptosis-related gene
   expression. Moreover, acute exposure to TCEP regulated MAPK-mediated
   transduction and regulation of GnRH signaling, thereby altering the
   expression of hypothalamic-pituitary-gonadal (HPG) axis-related genes.

Conclusions

   These findings revealed the endocrine disrupting effects of TCEP on
   turbot and identified biomarkers related to reproductive toxicity,
   providing early warning for the monitoring of healthy aquaculture.

Supplementary Information

   The online version contains supplementary material available at
   10.1186/s12864-024-11061-z.

   Keywords: Turbot, Tris (2-chloroethyl) phosphate (TCEP),
   Transcriptomics, Reproductive toxicity

Introduction

   Due to their adverse health effects, polybrominated diphenyl ethers
   (PBDEs) have been gradually banned in many countries, and
   organophosphorus flame retardants (OPFRs) have become the main
   substitutes [[36]1]. In recent years, the global production of OPFRs
   has increased rapidly, reaching an annual output value of 3 × 10^6 tons
   in 2018, 10 times more than in 2011 [[37]2]. As one of the most
   representative OPFRs, TCEP has been widely used in various industries,
   such as furniture, electronics, and building materials [[38]3]. TCEP is
   added to the material through physical mixing and has a high water
   solubility (25 °C, 7820 mg/L) and a low octanol-water partition
   coefficient (log Kow = 1.47), so high concentrations of TCEP have been
   frequently detected in various environmental media [[39]4]. During an
   investigation in 2023, a variety of organophosphate esters (OPEs) were
   detected in China’s Bohai Sea, with TCEP concentrations ranging from
   13.75 to 1721.29 ng/L [[40]5]. The highest concentration of TCEP
   (87.4 mg/L) was recorded in raw water from a Japanese sea-based
   wastewater disposal site [[41]6]. TCEP tends to occur with
   bioaccumulation and cascade amplification effects in the food chain
   [[42]7]. TCEP concentrations were detected in shrimp and crabs
   collected from aquafarms of the Beibu Gulf, China, with values ranging
   from 21.3 to 138 ng/g dw (mean 55 ng/g) [[43]8]. These data indicated
   that TCEP has become a pervasive emerging pollutant in the environment
   that may impact the health of aquatic organisms.

   In recent years, several studies have shown that TCEP exposure may
   result in neurotoxicity, developmental toxicity, reproductive toxicity,
   and even carcinogenesis in organisms [[44]9, [45]10]. For example, TCEP
   exposure significantly downregulated key genes and protein markers
   related to neurodevelopment in zebrafish [[46]11]. After 30 days of
   TCEP exposure, the survival rate, body weight, and specific growth rate
   of juvenile yellow catfish were found to be diminished [[47]12]. TCEP
   has also been shown to be an endocrine disrupting chemical [[48]13].
   TCEP exposure decreased testosterone (T) levels in mice, and the levels
   of genes involved in T synthesis were similarly downregulated [[49]14].
   Notably, the specific mechanisms of the neurotoxicity and reproductive
   toxicity of TCEP to fish and other aquatic organisms are still poorly
   understood.

   Turbot (Scophthalmus maximus) is a commercially significant aquaculture
   species in China. It is a highly nutritious fish that has been
   successfully farmed and bred in China since its introduction in 1992.
   By the end of 2019, the total cultivation area of turbot in China’s
   system demonstration zones was 6.0652 million m^3. In Europe, Spain is
   the main region for farmed turbot, with 7,450 tons produced in 2018
   [[50]15]. Owing to the accumulation of pollutants in the seawater, the
   turbot aquaculture industry is seriously threatened. Precocious sexual
   maturation in fish can increase disease susceptibility and health
   problems, in addition, gonadal development and maturation consume a
   considerable amount of energy, thereby reducing the overall growth rate
   and final body weight of fish and, ultimately leading to decreased
   production [[51]16]. For instance, Atlantic halibut males grow
   relatively slowly due to precocious sexual maturation [[52]17].
   Precocious European sea bass grew up to 18% less in weight than their
   counterparts during their second annual cycle of life [[53]18].
   However, the impact of new pollutants, including TCEP, on turbot
   culture has not been fully recognized in recent years.

   In teleost fish, the reproductive endocrine system is regulated by the
   hypothalamic-pituitary-gonadal (HPG) axis [[54]19]. The hypothalamus
   governs the release of luteinizing hormone (LH) and
   follicle-stimulating hormone (FSH) by secreting gonadotropin-releasing
   hormone (GnRH), thus controlling gonadal development and the secretion
   of sex hormones, estradiol (E2) and 11-ketotestosterone (11-KT)
   [[55]20]. LH and FSH are key regulators of the reproductive axis. They
   stimulate the gonads to produce a variety of sex steroid hormones at
   different stages, regulating reproductive functions [[56]21]. In
   addition, kisspeptin, an essential reproductive regulator encoded by
   the KISS gene, regulates the secretion of sex hormones and the sexual
   maturation process [[57]22].

   Transcriptomics, as a powerful tool for understanding the mechanism of
   toxicants, has been extensively employed to study gene expression and
   molecular pathways in aquatic stress responses [[58]23]. In the present
   study, turbot were exposed to TCEP for transcriptomic analysis of whole
   brain samples, and changes in plasma sex steroids (LH, FSH, E2, and
   11-KT) were measured, which aimed to reveal the potential mechanism of
   the neural and reproductive toxicity of TCEP exposure to turbot through
   transcriptomics.

Materials and methods

Chemicals and experimental fish domestication

   Tri (2-chloroethyl) phosphate (TCEP, CAS: 115-96-8; 99% purity) was
   purchased from Shanghai Macklin Biochemical Technology Co., Ltd. All
   reagents used in the exposure experiments were of analytical grade.

   Turbot was purchased from Shandong Kehe Marine High Technology Co.,
   LTD. (Weihai, China), which is the standard species with a consistent
   genetic profile, including 4-month-old juvenile turbot (body length
   17 ± 2 cm, weight 120 ± 5 g) in the early stage of sex differentiation
   and adult female turbot (body length 35 ± 1 cm, weight 1000 ± 200 g).
   Before the experiment, the fish were acclimatized to laboratory
   conditions for a period of 2 weeks in water tanks (72.2 cm × 52.5 cm ×
   43.3 cm, 110 L) with temperature 16 ± 2 °C, salinity 34.2 ± 0.2, pH
   7.8 ± 0.2, dissolved oxygen 7.5 ± 0.2 mg/L, and light cycle 14 L/10D
   (light/dark). Additionally, the fish were fed once a day at 15:00 with
   commercial feed provided by the farm at 3% of their body weight (i.e.,
   3.6 ± 0.15 g), and the seawater was entirely replaced every day.

TCEP exposure and sample collection

   Based on previous laboratory studies, the half-lethal concentration
   (LC50) of TCEP for 24 h treatment was 190.76 mg/L in juvenile turbots,
   and the stress response of turbot was more significant at 1/4LC50
   treatment [[59]24]. In addition, treatment concentrations exceeding
   environmental regulations were used in acute stress experiments to
   produce strong stress responses in organisms and to ensure that the
   results were meaningful. Therefore, the turbot were exposed to TCEP at
   an acute toxicity concentration of 1/4LC[50] (47.69 mg/L) for 2 h, 6 h,
   12 h, and 24 h as the experimental groups. while fish in clean seawater
   was used as the control group. To mitigate the influence of circadian
   rhythm, each group was sampled at 9:00 am. Fig. [60]S1 showed the
   experimental period, TCEP stress initiation time and sampling time of
   each group after domestication, which were represented by rectangles,
   triangles and pentagons respectively. Thirty individuals were included
   in each group, and three replicates were conducted for all groups. For
   each time endpoint, brain samples were collected from 3 individuals
   randomly selected from each replicate after anesthesia with a lethal
   dose of MS-222 (100 mg/L), and then frozen in liquid nitrogen for
   10 min and stored at − 80 °C. These tissue samples were collected for
   transcriptome analysis. All of the turbot collection and the anatomy
   experiments were conducted in accordance with the National Research
   Council’s Guide for the Care and Use of Laboratory Animals.

   In addition, 1-year-old turbots were exposed to 1.91 mg/L of TCEP. The
   experimental groups were treated with 1 d, 5 d, and 10 d, while the
   control group fish were kept in clean seawater (Fig. [61]S1B). Each
   group had three replicates with five fish per replicate. The
   experimental conditions were consistent with those during domestication
   except for TCEP concentration. During this period, the fish were fed
   commercial feed once daily at 15:00, totaling 3% of their body weight.
   Feeding was stopped 24 h before sampling. Blood samples were collected
   from the caudal vein via heparinized microcapillary tubes. Blood
   samples of three individuals per replicate were pooled as one sample
   and centrifuged at 4000 rpm for 20 min at 4 °C to separate supernatants
   for hormone quantification.

Hormonal measurement

   The levels of FSH, LH, E2, and 11-KT in the fish were determined using
   the corresponding commercial enzyme-linked immunosorbent assay (ELISA)
   kits (JL17630, [62]L13402, JL46284, and JL17632, respectively; Shanghai
   Jianglai Biotechnology Co., Ltd., China) according to the instructions.
   Serum from the control and TCEP exposure groups was used for testing
   and each experiment was replicated three times.

RNA extraction and transcriptome analysis

   All tissue samples for testing were packed in dry ice and sent to
   Novogene Biotech Co., Ltd. (Beijing, China). Total RNA was extracted
   from brain tissues of each group using TRIzol reagent (Invitrogen,
   Carlsbad, CA, United States). The integrity and concentration of RNA
   were determined by the Bioanalyzer 2100 system (Agilent Technologies,
   CA, USA). The NEBNext^® Ultra™ RNA Library Prep Kit for Illumina^® was
   used to prepare cDNA library. After library qualification, sequencing
   was performed on the Illumina NovaSeq 6000 (Illumina, USA), generating
   150 bp paired-end reads.

   After reads containing adapters, reads containing N bases, and
   low-quality reads were filtered out, the clean reads were obtained,
   which were mapped to the reference genome (GCF_022379125.1) obtained
   from NCBI for comparison using HISAT2 v2.0.5 [[63]25]. The FPKM
   (expected number of fragments per kilobase of transcript sequence per
   million base pairs sequenced) of each gene was used to assess the level
   of gene expression [[64]26]. Differentially expression analysis was
   performed using the DESeq2 R package v1.10.1 [[65]27]. Differentially
   expressed genes (DEGs) were screened with a threshold of adjusted P
   value < 0.05. GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes
   and Genomes) pathway enrichment analysis for DEGs were performed using
   Cluster Profiler (3.8.1).

Weighted gene co-expression network analysis (WGCNA)

   After filtering genes, the expression values of the screened genes
   (FPKM > 1) were used to construct co-expression modules comprising 15
   transcriptomes. The biological modules were classified according to the
   expression levels of genes and the similarity of module eigengenes
   (ME). Pearson correlation coefficients of relationships between the
   module and the sample trait were calculated according to the ME value
   of the module. Modules with Pearson correlation coefficients > 0.5 and
   P < 0.05 were considered to be associated with TCEP exposure, and GO
   and KEGG analyses were performed to clarify the biological functions of
   these response modules [[66]28].

Reproductive related gene analysis

   The total RNA used in qRT-PCR assays was consistent with that for
   transcriptome sequencing. And reverse transcription was performed using
   PrimeScript RT^® reagent kit with gDNA Eraser (Takara, China), and then
   test the expression of target genes (Gt, GnRH, FSH, LH, KISS, and
   KISSr) using qPCR SYBR Green Master Mix kit (Takara, Japan) in a
   LightCycler^® 96 Instrument (Roche, USA) with the 20-µl reaction system
   including 10 µl PCR Premix (SYBR), 0.5 µl PCR forward primer, 0.5 µl
   PCR reverse primer, 2.0 µl cDNA, and 7.0 µl dH[2]O. Specific primers
   and annealing temperature were shown in Table [67]S1. The templates
   were amplified in triplicate with 35 cycles of 95 ℃ for 10 s, annealing
   for 30 s, and 72 ℃ for 30 s. As a reference gene, the β-actin was used
   to normalize the cycle threshold (Ct) value. To ensure the accuracy and
   reliability of the results, standard samples of different
   concentrations were used to draw standard curves. By comparing with the
   Ct value of the sample, the relative expression of the target gene was
   calculated by the comparative Ct (2 ^−∆∆Ct) method [[68]29].

Statistical data analysis

   Statistical analysis and visualization were performed using GraphPad
   Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). The
   mean ± standard deviation (SD) was used to express experimental data.
   Hormone levels and relative gene expression at different time points
   were examined using one-way analysis of variance (ANOVA), after
   evaluating the normality and homogeneity of variance. Comparisons
   between the TCEP group and the control group were specifically assessed
   using the Dunnett test to evaluate significant differences. The
   significance levels were denoted by asterisks (*) (∗P < 0.05,
   ∗∗P < 0.01).

Results

Effect of TCEP on reproductive hormone content

   The results showed that with increasing TCEP exposure time, the
   hormonal contents of 1-year-old turbot plasma changed (Fig. [69]1).
   Compared to the control, the contents of FSH, LH, E2, and 11-KT showed
   a decreasing trend. After 10 days, the lowest values occurred with a
   decrease of 3–7%. The decrease in 11-kT was the smallest. Therefore,
   continuous exposure to low concentrations of TCEP caused a gradual
   decrease in reproductive hormones in turbot.

Fig. 1.

   [70]Fig. 1
   [71]Open in a new tab

   Changes in reproductive hormones levels after exposure to TCEP
   (1.91 mg/L) exposure for 1, 5, and 10 days. (A-D) The levels of
   luteinizing hormone (LH), follicle stimulating hormone (FSH), estradiol
   (E2), and 11-ketone testosterone (11-KT). Values represent the
   mean ± standard deviation (n = 3). Asterisks denote significant
   differences between TCEP and control (∗p < 0.05, ∗∗p < 0.01)

Library construction and sequencing analysis

   We evaluated the quality of transcriptome sequencing data from 15
   samples (Table [72]S2). After quality trimming, 653,379,440 clean reads
   were generated from 683,482,810 raw reads, with an error rate of less
   than 0.01%. The Q20 and Q30 values of each sample were no less than
   98.39% and 95.66%, respectively, and the GC content ranged from 46.11
   to 50.63%. A comparison of the clean reads with the reference genome
   revealed that the concordance rate of each sample was greater than
   83.95% (Table [73]S3). These date suggested that the transcriptome data
   were of high quality.

Identification and analysis of DEGs

   We further evaluated the overall gene expression profile by examining
   DEGs associated with TCEP exposure in whole brain tissue of turbot.
   There were 2,128 (1,075 up- and 1,053 down-regulated), 3,307 (1,599 up-
   and 1,708 down-regulated), 2,069 (1,014 up- and 1,055 down-regulated),
   and 3,571 (1,819 up- and 1,752 down-regulated) DEGs in the N_2h vs.
   N_Ctrl, N_6h vs. N_Ctrl, N_12h vs. N_Ctrl, and N_24h vs. N_Ctrl groups,
   respectively (Fig. [74]2A), which were also illustrated in volcano
   plots (Fig. [75]2E-H). Venn diagram showed that 1157 DEGs were shared
   among the four TCEP-exposed groups (Fig. [76]2B). The square of
   Pearson’s correlation coefficient (R2) exceeded 0.961, indicating high
   similarity in expression patterns between samples (Fig. [77]2C).
   Principal component analysis (PCA) plot revealed complex sample
   composition relationships for two eigenvalues (PC 1:30.13%, PC
   2:11.73%) (Fig. [78]2D). The above analysis indicated that a large
   number of genes responsded to TCEP stress.

Fig. 2.

   [79]Fig. 2
   [80]Open in a new tab

   Transcriptome analysis of turbot exposed to nitrate. (A) DEGs between
   CK and different treatment groups. (B) Venn diagram showed the number
   of shared and unique DEGs between different groups. (C) Cluster heat
   map analysis. (D) Principal component analysis. (E-H) represented
   volcano plots of the DEGs between Ctrl and different treatment groups
   (N_2h, N_6h, N_12h, and N_24h)

   GO functional enrichment analysis was conducted on the DEGs obtained
   from different stress groups. Thirty enriched GO terms were displayed,
   including 10 items each for biological process (BP), cellular component
   (CC), and molecular function (MF) (Fig. [81]S2). For example, cell
   signal transduction, protein biosynthesis and folding, nucleic acid
   metabolism, and organelle function regulation were enriched in BP,
   extracellular structure and space, cell membrane-related components,
   and organelles in CC, and gene transcription regulation, protein
   binding and catalytic activity, ion transport, and biomolecular binding
   and catalytic activity in MF.

   To gain a better understanding of the cellular metabolic pathways
   involved in DEGs, the top 20 enriched KEGG pathways with the most
   abundant DEGs for four exposure groups were displayed (Fig. [82]3A-D).
   In addition to some metabolism-related pathways, “Oxidative stress,
   oxidative damage and apoptosis” and “Signal transduction” were common
   enrichment pathways, suggesting that acute exposure to TCEP could
   initially induce the responses of these pathways in turbot. In
   addition, the metabolic pathways associated with the endocrine system
   and oxidative stress were further investigated in this study
   (Table [83]1).

Fig. 3.

   [84]Fig. 3
   [85]Open in a new tab

   Enrichment analysis of differentially expressed genes (DEGs) in KEGG
   pathways. (A-D) The top 20 pathways after 2, 6, 12, and 24 h of acute
   TCEP exposure, respectively. (E, F) Heat maps of genes related to
   oxidative phosphorylation and apoptosis pathway

Table 1.

   Enrichment profiles of pathways related to the endocrine system and
   oxidative stress under TCEP exposure
   Pathway N_2h N_6h N_12h N_24h
   p value Ratio (up-regulated DEGs/total DEGs) p value Ratio
   (up-regulated DEGs/total DEGs) p value Ratio (up-regulated DEGs/total
   DEGs) p value Ratio (up-regulated DEGs/total DEGs)
   Endocrine system GnRH signaling pathway 0.304 8/15 0.168 21/25 0.226
   8/15 0.001 22/39
   Steroid biosynthesis 0.007 3/7 0.533 0/4 0.019 1/6 4.7E-4 1/12
   PPAR signaling pathway 0.001 13/19 0.559 5/14 0.305 3/10 5.49E-4 11/30
   Notch signaling pathway 0.036 7/14 0.995 3/6 0.385 6/9 0.630 9/15
   Adipocytokine signaling pathway 0.055 4/14 0.173 5/18 0.425 1/9 0.036
   10/24
   Oxidative stress Oxidative phosphorylation 0.323 14/17 0.042 14/32
   0.999 2/5 5.51E-13 1/66
   Ferroptosis 0.024 4/11 0.112 6/13 0.162 4/8 0.001 7/21
   Apoptosis 0.038 13/26 0.008 17/42 0.884 3/13 2.85E-4 21/54
   Phagosome 0.002 19/30 0.443 9/29 0.277 4/19 9.6E-5 13/54
   MAPK signaling pathway 0.133 25/47 0.001 52/87 0.006 19/53 0.230 52/82
   FoxO signaling pathway 0.387 8/19 0.007 24/41 0.359 6/18 0.006 27/47
   [86]Open in a new tab

WGCNA analysis under TCEP exposure

   WGCNA was performed to identify gene co-expression networks responding
   to TCEP stress. Based on 14,543 genes with FPKM > 1, a weighted gene
   co-expression network was constructed with an empirical soft threshold
   of 18 and an average connectivity of 36 (Fig. [87]S3A, B). On the basis
   of the similarity of the gene expression levels, a gene clustering tree
   was constructed, and 17 different co-expression modules were obtained
   (Fig. [88]4A). We used module eigengenes (MEs) to represent the
   expression level of genes in each module, and analyzed the interactions
   between these modules (Fig. [89]4B). The results of the module-trait
   relationship indicated that six modules were significantly associated
   with TCEP exposure traits, including purple and green corresponding to
   TCEP for 2 h, turquoise, magenta, and tan corresponding to TCEP for
   12 h, and midnight blue corresponding to TCEP for 24 h. The ME. pink
   module had the strongest correlation with the Ctrl group (Fig. [90]4C).
   The top 20 DEGs with weight values in the seven modules were shown in
   Table [91]S4. DEGs in the above seven modules were subjected to KEGG
   enrichment analysis (Fig. [92]4D), and the enrichment results were
   consistent with those of four TCEP-treated groups.

Fig. 4.

   [93]Fig. 4
   [94]Open in a new tab

   Weighted gene co-expression network analysis (WGCNA) of acute TCEP
   stress. (A) The hierarchical clustering tree represents the
   co-expressed modules determined by WGCNA. (B) Heatmap of eigengene
   adjacency. (C) Heatmap of correlation between traits and modules. (D)
   Enrichment pathway of seven modules significantly associated with
   TCEP-exposure traits

Expression of genes associated with reproduction

   The changes in the expression of reproductive-related genes in turbot
   after TCEP exposure were shown in Fig. [95]5. With prolonged TCEP
   stress, the expression levels of Gt and GnRH genes encoding
   gonadotropin-releasing hormone generally increased and then decreased.
   At 6 h, the response was 50 times and 150 times that of the control
   group, respectively. The highest levels of LH and FSH also occurred in
   the 6 h treatment groups. The expression of FSH gene responding to TCEP
   was lower than that of LH. The expression of KISS and KISSr in the
   treatment groups changed significantly, and the expression of KISS
   decreased significantly at 12 h and 24 h.

Fig. 5.

   [96]Fig. 5
   [97]Open in a new tab

   (A-F) The relative expression levels of reproductive genes (Gt, GnRH,
   LH, FSH, KISS, and KISSr). * represents p < 0.05 and ** represent
   p < 0.01, indicating significant differences between the exposure
   groups and the control group

Discussion

   TCEP is an environmental endocrine disruptor, and due to its widespread
   presence in the natural environment, an increasing number of studies
   have evaluated its toxic effects. However, limited attention has been
   paid to its neurological and reproductive effects on aquatic organisms.
   With the increasing use of TCEP, it has been frequently detected in
   water environments because of its high water solubility [[98]30,
   [99]31]. Therefore, TCEP pollution and its toxic effects on aquatic
   organisms cannot be overlooked. According to the physical properties of
   TCEP and the results of previous experiments, 1/4LC[50] (49.67 mg/L)
   TCEP was used as the stress condition. Through high-throughput
   sequencing technology and WGCNA, 2069–3571 DEGs (relatively more DEGs
   in the N_6h and N_24h groups) and seven modules related to TCEP stress
   in turbot brains were screened to investigate the molecular mechanism
   of the turbot response to TCEP exposure.

Effects of TCEP exposure on neurotoxicity

   Oxidative stress is the most common mechanism by which aquatic
   organisms respond to various environmental pollutants [[100]32]. TCEP
   exposure can lead to the generation of reactive oxygen species (ROS),
   thereby causing an imbalance in oxidation-antioxidant processes and
   undermining the cellular structure and function [[101]33, [102]34].
   Life-cycle exposure to TCEP activated the Nrf2-Keap1 antioxidant
   pathway in response to TCEP-induced oxidative stress in zebrafish gills
   [[103]35]. KEGG analysis showed that several significantly enriched
   pathways were associated with neuropathy, particularly oxidative
   phosphorylation pathway, and similarly, GO term also revealed that TCEP
   affected cell signaling and redox processes in mitochondria, which were
   critical for mitochondrial function and neurodevelopment. Oxidative
   phosphorylation normally occurs in the mitochondria via the respiratory
   chain and ATP synthase, and it is one of the major cellular pathways
   used to generate adenosine triphosphate (ATP). The expression of genes
   encoding mitochondrial electron transport chain components, including
   NADH dehydrogenase (complex I), cytochrome c reductase (complex III),
   cytochrome c oxidase (complex IV), and ATP synthase (complex V), was
   altered. Nduf encodes multiple subunits of complex I, a 45-subunit
   enzyme complex that uses non-covalently bound prosthetic groups to
   facilitate the initial step of the electron transport chain [[104]36].
   atp5f1c, atp5f1d, atp5l, and atp5mf genes encode subunits of complex V,
   which work together to synthesize ATP at the inner mitochondrial
   membrane to provide energy required for the cell [[105]37]. The
   downregulation of related genes led to impaired function of the
   electron transport chain, eventually resulting in neuronal damage, cell
   apoptosis, and even reproductive toxicity [[106]38]. Multiple studies
   have shown that exposure to certain refractory and readily accumulating
   pollutants during the critical developmental period of the brain may
   permanently affect some signaling pathways in the nervous system
   [[107]39]. Our findings indicated that impaired mitochondrial function
   with TCEP exposure may result in an inability to meet the high energy
   demand of brain neurons, potentially leading to impaired brain cell
   function or cell death. Apoptosis is an important biological mechanism
   for the body’s resistance to pathogen infection or environmental
   stress, which helps eliminate damaged cells and maintains tissue
   homeostasis [[108]40]. In this study, several genes associated with
   apoptosis, such as c-Jun, caspase-7, and ctsl.1, showed significant
   expression after TCEP stress. When an apoptotic signal is received from
   a transmembrane receptor, the extrinsic pathway is activated, which
   leads to the cascade activation of caspase-7 [[109]41]. Caspase-7 is
   involved in the execution phase of apoptosis, which can be induced by
   activating other apoptosis-related proteins, such as nucleases. ATF4B
   is a transcription factor that can influence the activation and
   execution of the apoptotic pathway, thereby promoting cell death. In
   addition, the expression of c-Jun was upregulated, which also promotes
   cell apoptosis [[110]42]. The expression levels of pro-apoptotic genes
   were increased, thus inducing apoptosis. This inference is consistent
   with the adverse effects of TCEP on neurodevelopment [[111]11]. We
   concluded that after TCEP stress, the energy metabolism of fish brain
   cells was blocked and accompanied by apoptosis, which may affect the
   nervous system and further affect the regulation of reproduction.

Effects of TCEP exposure on reproductive toxicity

Toxicity of TCEP on gonadotropins and sex hormones

   The hypothalamic-pituitary-gonadal axis is a complex endocrine system
   that plays a key role in the development and regulation of the
   reproductive system in fish and other animals. The main regulators of
   the HPG axis are several hormones, such as GnRH, LH, FSH, E2,11-KT, and
   their specific receptors [[112]43]. During the gonadal development
   cycle, the GnRH level changes continuously. The pituitary gland
   synthesizes and releases gonadotropins (GtHs) into the blood in
   response to these changes. GnRH has been found to regulate the
   secretion and synthesis of GtHs in salmonids and rainbow trout
   (Oncorhynchus mykiss) [[113]44]. GtHs are essential regulators of HPG
   axis. Initially, only one type of GtH was thought to act in fish, but
   later two types of GtH were isolated from the pituitary glands of
   various fish, such as salmonids [[114]45], zebrafish [[115]46], and
   goldfish [[116]47]. Thus, LH and FSH, formerly called GtH I and GtH II,
   respectively, are the two major gonadotropins in teleost fish, which
   were present in turbot. 1-year-old female turbot whose oocytes are
   usually in early growth or early maturation. At this point, the oocyte
   has begun to grow and accumulate nutrients in preparation for
   subsequent maturation and ovulation. In our study, we observed that the
   levels of FSH and LH were decreased following exposure to TCEP. FSH
   plays a significant role in the vitelline production and gametogenesis
   stages of fish gonad development [[117]48]. LH plays an important role
   in germ cell maturation and release [[118]49]. Our results were
   consistent with those previously shown for other endocrine disruptors.
   Bisphenol A (BPA) exposure decreased LH and FSH levels and reduced
   sperm quality in Wistar rats [[119]50]. Exposure of zebrafish to TBBPA
   for 40 days resulted in significantly lower levels of LH and FSH
   [[120]20]. Sex steroid hormones are divided into estrogen, androgen,
   and progesterone, and all types of sex steroid hormones are synthesized
   de novo from the precursor cholesterol, and the synthesis process is a
   cascade reaction involving the nervous and endocrine systems [[121]51].
   In our study, the levels of E2 and 11-KT decreased with the duration of
   TCEP stress, which might be related to the fact that TCEP may cause
   damage to nerve cells. The continuous reduction in E2 and 11-KT would
   directly affect the normal maturation and secretion of eggs, delay the
   production of young turbots, and cause great hindrance to the breeding
   work of turbot. TCEP induced pathological changes and inhibited gonadal
   maturation by inhibiting the plasma sex hormone concentration in
   zebrafish [[122]52]. A decrease in FSH and LH might lead to a decrease
   in E2 and 11-KT, resulting in lower sperm quality and quantity and an
   increase in sperm malformations. Lifetime exposure to TCEP interfered
   with biosynthesis of steroid hormones through multiple pathways, and
   impaired testicular development and spermatogenesis of zebrafish
   [[123]30]. In vitro, TCEP stress increased estradiol and testosterone
   concentrations in human adrenal cortical cancer cells [[124]53],
   whereas in vivo, TCEP stress reduced hormone levels in the testes of
   mice [[125]54]. In our results, chronic exposure to TCEP induced the
   contents of LH, FSH, E2, and 11-KT in turbot, which was consistent with
   the findings in mammals. It is possible that under the environment of
   unlimited growth in vitro, the hormone content may continue to rise,
   while the feedback regulation mechanism in vivo will reduce the hormone
   content. These results provide evidence for specific reproductive
   toxicity studies of TCEP.

Effects of TCEP on reproductive pathways

   TCEP may induce reproductive toxicity and cytotoxicity in turbot by
   affecting signaling to modulate the regulation of gonadotropins by
   GnRH, and the genes involved in the regulation were altered
   (Fig. [126]6). The GnRH signaling pathway is a key system that
   regulates reproductive function. After GnRH binds to its receptor
   (GnRHR), it can activate a variety of downstream pathways, such as
   Ca^2+ signaling pathway, cAMP signaling pathway, and MAPK signaling
   pathway, which affect the function of the reproductive system
   [[127]55]. GnRHR couples to Gs, activating adenylate cyclase (AC) and
   increasing cyclic adenosine monophosphate (cAMP) levels, which
   activates protein kinase A (PKA). PKA phosphorylates CREB, enabling its
   binding to cAMP response elements (CRE) in DNA to regulate gene
   transcription [[128]56]. gnas is a gene encoding the alpha subunit of
   Gs that interacts with other signaling molecules to regulate signaling
   pathways within cells [[129]57]. The conversion of ATP to cAMP is
   catalyzed by AC, adcy2a, and adcy8 genes encode this enzyme. cAMP binds
   to PKA, activating it to phosphorylate various substrate proteins, thus
   regulating their activity. prkacab, prkacba, and prkacbb encode the PKA
   complex. Ethylhexyl salicylate induced abnormal melanin in F0 via cAMP,
   MAPK, and Wnt signaling pathways. with the UV-regulated cAMP signaling
   pathway being the main pathway [[130]58]. In the mouse LβT2 cell line,
   exogenous SN activates PKA and protein kinase C (PKC)-dependent
   signaling pathways, thereby promoting LH expression [[131]59]. In
   addition, mitogen-activated protein kinase (MAPK) and Ca/calmodulin
   dependent kinase II (Ca/CaMKII) pathway are also involved in
   GnRH-induced GTH subunit gene expression [[132]60]. In this pathway,
   mapk11, and mapk13 encoding p38MAPK, calm2a and calm3a encoding
   calmodulin, and LOC118287871 and camk2b1 encoding
   Ca^2+/calmodulin-dependent protein kinase (CaMK) were generally
   upregulated and then downregulated with TCEP stress. In a goldfish
   study, decreased levels of PKC resulted in decreased gonadotropin
   secretion in response to GnRH stimulation [[133]61]. CaMK II plays a
   key role in ERK activation at slow pulse frequencies, regulating the
   expression of LHβ and FSHB genes [[134]62]. Altered expression of genes
   in the GnRH signaling pathway led to a rapid increase in the expression
   of genes related to reproduction (GnRH, LH, and FSH) in turbot, which
   led to an increase in the corresponding hormone expression, resulting
   in advanced sexual maturity. However, after 24 h of TCEP stress, the
   expression of related genes decreased, which might be caused by the
   failure of turbot to recover in time after the treatment time exceeded
   12 h. Combined with the effects of TCEP on the nervous system, it
   indicated that TCEP exerted neuroendocrine disturbance on turbot.
   Studies have shown that the regulation mechanism of the HPG axis was
   disrupted, and sex hormone secretion was severely disrupted in adult
   zebrafish after exposure to OPEs, which eventually led to the
   destruction of fish reproductive ability, which was similar to the
   effect of TCEP on turbot.

Fig. 6.

   [135]Fig. 6
   [136]Open in a new tab

   Expression changes of genes related to GnRH signaling pathway of turbot
   upon TCEP exposure

Effects of TCEP on reproductive gene expression

   Genes on the HPG axis were also examined in this study, and the results
   not only validated the accuracy of the transcriptomic results, but also
   indicated that TCEP may cause reproductive toxicity by affecting the
   expression levels of Gt, GnRH, LH, and FSH in turbot. GnRH encodes
   gonadotropin-releasing hormone, which regulates the synthesis and
   release of gonadotropins in the anterior pituitary. FSH and LH encode
   two types of gonadotropins respectively, which regulate the function of
   the reproductive system and play important roles in follicle
   development, sperm formation, and sex hormone synthesis. In our study,
   the expression levels of reproductive genes were up-regulated and
   reached a peak at 6 h of TCEP exposure, and then decreased, which was
   consistent with the transcriptome levels. The data supported that the
   contents of reproductive hormones (FSH, LH, E2, and 11-KT) decreased
   with long-term TCEP stress. Kisspeptin, a class of neuropeptides
   encoded by the Kiss1, is produced mainly by specific hypothalamic
   neuronal clusters, whose expression is regulated by GPR54 [[137]63].
   This receptor is associated with a variety of hormonal signaling
   pathways, including those involved in sexual maturation and
   reproduction [[138]64]. Based on our results, it was speculated that
   TCEP exposure influenced the change in hormone levels by altering the
   expression of KISS and HPG axis-related genes, thereby regulating the
   timing of egg maturation.

WGCNA analysis under TCEP stress

   While many studies have investigated gene expression under
   environmental stress, few have done so by constructing gene
   co-expression networks. In our WGCNA, 17 co-expression network modules
   were obtained. Six modules (purple, green, turquoise, magenta, tan, and
   midnight blue) were significantly associated with TCEP exposure traits,
   and one (pink) was significantly correlated in the Ctrl group, which
   suggested that ME. pink module genes play key roles in maintaining
   normal physiological functions under stress. KEGG enrichment analysis
   of the top 20 weighted genes showed that oxidative stress and
   reproduction were significantly enriched after TCEP exposure, including
   oxidative phosphorylation, phagosome, ferroptosis, GnRH signaling
   pathway, etc., which were also enriched in the four treatment groups.
   The enriched genes related to oxidative phosphorylation, such as
   LO118302460, atp6v1ba, and ndufa12, were considered key genes that play
   important roles in encoding the mitochondrial respiratory chain
   complex. Once mitochondrial dysfunction occurs, it may lead to energy
   metabolism disorder, which in turn affects the normal physiological
   function and health of the organism. TCEP stress caused decreased
   transcript levels of these key genes, indicating that oxidative
   phosphorylation pathway was closely related to stress, and turbots were
   in an unhealthy state. Moreover, phagocytosis is an actin-based cell
   internalization process that removes host cells undergoing apoptosis or
   necrosis, the activation of which is an important immune mechanism
   [[139]65, [140]66]. Our study showed that some phagosome genes were
   up-regulated, which may enhance the phagocytosis activity of cells and
   clear damaged cells (Fig. [141]S4). These findings proved that
   insufficient energy metabolism led to cell damage and triggered
   phagocytosis of immune cells. Ferroptosis appeared to be sensitive to
   TCEP exposure, as it was enriched in all the groups and the seven
   modules group. Experimental compounds and some clinical drugs have been
   reported to induce ferroptosis in normal and cancer cells, which has
   become a hot topic in life science research [[142]67]. Low temperature
   stress caused renal dysfunction and the downregulation of
   immune-related pathways as well as iron deficiency in tilapia
   [[143]68]. Iron-based nanomaterials enlarged the ventricles or atria of
   zebrafish, leading to reproductive toxicity [[144]69]. WGCNA analysis
   found that TCEP exposure led to ferroptosis, but the exact mechanism
   remains unclear, which should be further investigated in the future.

Conclusions

   In this study, we conducted a comprehensive analysis of the effects of
   TCEP stress on the turbot at different time points at the nucleic acid
   and protein levels using the determination of hormone content and
   transcriptomics analysis. Under TCEP stress, the expression of GnRH,
   LH, FSH, KISS, and KISSr genes was altered, whereas the levels of four
   reproductive hormones (LH, FSH, E2, and 11-KT) levels were
   significantly decreased. These changes may affect the earlier sexual
   maturity of turbot, and may also cause the inability of adult fish to
   release eggs properly and reproduce the second generation of fish,
   which will lead to a serious decline in production and cause economic
   losses. Transcription profile analysis showed that DEGs were enriched
   in pathways related to oxidative stress and reproductive regulation,
   including oxidative phosphorylation and apoptosis, GnRH signaling
   pathway and MAPK signaling pathway, respectively. Furthermore, WGCNA
   analysis revealed the modules and key pathways, among which oxidative
   phosphorylation was the most significant, while we also found that
   phagosome and ferroptosis pathways were enriched. Our results revealed
   that TCEP affected mitochondrial function and energy metabolism,
   indicating that TCEP may cause long-term toxicity, but the specific
   mechanism needs further investigation. Our study may contribute to the
   understanding of the neuroendocrine-disrupting effect of TCEP and
   provide guidance for turbot cultivation.

Electronic supplementary material

   Below is the link to the electronic supplementary material.
   [145]Supplementary Material 1^ (42.5KB, doc)
   [146]Supplementary Material 2^ (37.5KB, doc)
   [147]Supplementary Material 3^ (97KB, xls)
   [148]Supplementary Material 4^ (15.7KB, docx)
   [149]Supplementary Material 5^ (5.3MB, tif)
   [150]Supplementary Material 6^ (4.1MB, tif)
   [151]Supplementary Material 7^ (16.5MB, tif)
   [152]Supplementary Material 8^ (106.7MB, tif)
   [153]Supplementary Material 9^ (3MB, tif)

Acknowledgements