Cortisol signaling and stress-induced gene expressions in response to copper toxicity through heat stress during zebrash embryogenesis

Climate change is leading to an increase in temperatures, which has a stressful impact on the aquatic environment. Cortisol signaling is involved in enhancing metabolic processes such as anti-oxidation, immune defense, and osmoregulation, under stress conditions in sh. The present study aimed at evaluating the effects of copper (Cu) toxicity along with an increase in temperature during zebrash embryogenesis, based on the transcriptional responses of cortisol and stress-related genes. A decreased survival rate was observed following combined exposure to high temperature and Cu. Heart rates of zebrash embryos signicantly increased only during heat stress. An abnormal morphology was induced by exposure to a combination of Cu and heat stress. Furthermore, heat stress also triggered Cu-induced intracellular reactive oxygen species production with upregulation of superoxide dismutase (SOD) and glutathione s-transferase (GST) and cell death with modied expressions of p53 and B-cell lymphoma-2 (Bcl-2) in the zebrash embryo. Finally, increased cortisol level and altered expressions of cortisol-signaling genes were observed following exposure to Cu and high temperature. These results highlight that the realistic exposure to combined stressors disturbs cortisol-related defense pathways as well as the stress-induced processes of anti-oxidation and cell death in sh. Cu induced the elevation of cortisol levels in zebrash; those of previous studies on sh 12,13 . The induction of cortisol levels is intensied by combined exposure to high temperature and Cu stressors. Increase in cortisol levels can be correlated to the hormonal defense responses to thermal stress and copper toxicity as a sensitive indicator of in sh. Our results showed that the expression of genes related to the cortisol process is affected by exposure to high temperature and Cu singly or in combination. cortisol levels may also be due to the inhibition of cortisol catabolism and excretion, evidenced by the downregulated expression of Hsd11b2 and Hsd20b2. Hsd11b2 a crucial role in


Introduction
Water temperatures are expected to increase worldwide due to global climate change. The aquatic environments are undergoing unprecedented changes, including rising temperatures, alterations of chemical properties, and changing susceptibility of organisms 1,2 . Cortisol, the key corticosteroid in sh, plays a pivotal role in stress response in physiological processes including growth, reproduction, metabolic pathway, immune defense, and osmoregulation [3][4][5] . The stress response involves broad changes, from a molecular to an individual level, set to reduce potential toxic effects of the stressor and maintain homeostasis 6 . Plasma cortisol levels are elevated during thermal stress induced by increasing temperature in juvenile Senegalese sole (Solea senegalensis) and Atlantic cod (Gadus morhua L.) 7,8 . Stressful conditions caused by elevated temperature induce cortisol binding to glucocorticoid receptor (GR) and serious alterations of the GR-complex in sh 9,10 .
Copper (Cu), an essential trace metal, serves as a cofactor in metalloproteins induced in crucial cellular processes in aerobic metabolism. However, elevated Cu levels induce toxic effects in sh through the production of the reactive oxygen species (ROS) in redox-cycling 11 . Cu exposure triggers alteration of the cortisol level in common carp, Cyprinus carpio and in rainbow trout, Oncorhynchus mykiss 12,13 . The change in cortisol level may be a defense response to reduce the chronic stress of Cu exposure, and not due to endocrine disruption 13 . Stressor combinations that re ect a realistic environment generally evoke complex responses in sh, but the underlying mechanisms remain largely unknown. Heat-and Cuinduced stress signi cantly increased expressions of DNA methyltransferases and heat-shock protein 70 (hsp70) in zebra sh. However, the temperature-dependent elevation of Cu toxicity is not related to the energy metabolic pathway, although response to Cu showed an enhancement in hepatic aerobic metabolism related to the energy-demanding process of metal detoxi cation in the killi sh Poecilia vivipara 14 .
In order to determine whether thermal stress induced by increasing temperature can booster Cu toxicity in an aquatic environment, we investigated the physiological phenotypes, molecular responses of cortisol signaling, and stress-induced gene expressions to various Cu concentrations and heat stress exposures during zebra sh embryogenesis. This study aimed to verify the knowledge on the enhanced sensitivity of stress responsive indicators, including cortisol signaling mechanism, using transcriptional approaches in sh exposed to heat and Cu stressors.

Results
Biological responses to combined stressors. Figure 1 shows that exposure to 0.1 mg L − 1 of Cu at 26°C, decreased the survival rate to 70% at 2 days post fertilization (dpf), this survival rate was maintained till 7 dpf. Increasing the temperature (34°C) increased Cu toxicity at concentrations over 0.03 mg L − 1 , thus affecting the survival rate of the zebra sh embryos (10% at 3 dpf, Fig. 1B). However, heart rates were not different at combined exposures to Cu and heat compared to the control, although they signi cantly increased on exposure to elevated temperature (Fig. 1C). Following changes in the temperature, there was no difference in the size of yolk sac edema between the control and Cu-treated groups (Fig. 1D). The incidence of malformation (screened on the basis of curved body shape) increased on combined exposure to all Cu concentrations and heat stress (Fig. 1E).
ROS production and cell death in live zebra sh embryos. At 26°C, a signi cant increase in intracellular ROS production was observed at 0.007 and 0.01 mg L − 1 Cu exposure in the live zebra sh embryos. Increasing the temperature signi cantly boosted the induction of intracellular ROS production at all Cu concentrations (Fig. 2). In addition, the expression of the antioxidant genes, superoxide dismutase (SOD) and glutathione s-transferase (GST), was signi cantly increased in response to Cu exposure at an elevated temperature. Cu-induced cell death caused by increasing the temperature was observed in live zebra sh by measuring the uorescence intensity using acridine orange (Fig. 3). The mRNA of apoptosisrelated genes, p53 and B-cell lymphoma-2 (Bcl-2), were expressed at a relatively high concentration of Cu (0.01 mg L − 1 ). Thermal stress also boosted Cu-induced effects in the antioxidant defense mechanism and apoptotic process in zebra sh.
Cortisol level and cortisol signaling expression. The cortisol levels in 96 hours post fertilization (hpf) larvae following exposure to 0.003, 0.007, and 0.01 mg L − 1 Cu at either 26°C or 34°C is shown in Fig. 4A. The cortisol levels were signi cantly increased on combined exposure to Cu and heat stress than on exposure to only Cu. The expression pro les of the 14 cortisol-and stress signaling-related genes are depicted in Fig. 4B. Elevated temperature at 34°C induced mRNA expression of Fdx1b, GST, and SOD. Combination of heat and Cu exposure signi cantly increased the transcriptional expression of Fdx1b, GR, Cyp2k22, GST, Crhb, SOD, Foxi3a, and Cyp11c1, whereas mRNA expression of Hsd20b2, Hsd11b2, Cyp11a1, Cyp11b1, and Bcl-2 decreased on exposure to Cu and elevated temperature. Expression of p53 was upregulated at 0.007 and 0.01 mg L − 1 Cu at 34°C. The most signi cant upregulation of GR and SOD was observed under exposure to 0.01 mg L − 1 Cu and heat stress (P < 0.01).

Discussion
In the present study, we investigated the correlation between sensitivity to Cu toxicity enhanced by increased temperature and the consequent disturbance in the cortisol signaling process during sh embryogenesis. Dorts et al., 2016 15 reported evidence of epigenetic modi cation in zebra sh upon exposure to heat and Cu during reprogramming of DNA methylation. Overall, in this study, the toxic stress of Cu induced by high temperature resulted in changes in the expression of cortisol signaling genes as well as antioxidant and apoptosis-related genes during zebra sh embryo development.
Fishes are frequently exposed to multiple stressors in combination introduced by intensive human activities or from natural sources that lead to the discharge of heavy metals into the aquatic environment 15,16 . Thermal stress induced by an increase in temperature greatly alters the effects of Cu pollutants 11,17,18 . The interaction of Cu and thermal stress modulated Cu-induced bioenergetic disturbances, including impairment of oxidative phosphorylation, by inhibiting the electron transport chain in rainbow trout, Oncorhynchus mykiss 11,19 . Elevated temperatures accelerated the toxic effects of Cu through metal accumulation and elevated oxidative stress-related processes in the killi sh Poecilia vivipara 20 . In our study, heat stress boosted ROS production induced by Cu toxicity, also including the increased expression of antioxidant genes such as SOD and GST. Both stressors, high temperature and Cu, triggered cell death in zebra sh via the upregulation of the apoptosis-related gene p53 and downregulation of the anti-apoptotic gene Bcl-2. Several studies have reported Cu-induced apoptosis in sh [22][23][24] . In the present study, we demonstrated that high temperature boosted Cu toxicity in apoptosis during zebra sh embryogenesis. Cell death in live zebra sh is likely to have been elicited by the production of ROS, similar to results observed for tilapia (Oreochromis mossambicus) 25 .
Cortisol signaling regulates a broad range of metabolic and physiological process 26 . Enhanced release of cortisol in sh is re ected as hormonal stress responses to exposure to heavy metals 27 . Stressful conditions caused by elevated temperature induce cortisol binding to GR and serious alterations of the GR-complex in sh 10,28 . Plasma cortisol levels were elevated by thermal stress induced by increasing temperature in juvenile Senegalese sole (Solea senegalensis) and Atlantic cod (Gadus morhua L.) 8, 29 . In the present study, Cu exposure induced the elevation of cortisol levels in zebra sh; this result is in agreement with those of previous studies on sh 12,13 . The induction of cortisol levels is intensi ed by combined exposure to high temperature and Cu stressors. Increase in cortisol levels can be correlated to the hormonal defense responses to thermal stress and copper toxicity as a sensitive indicator of environmental stress in sh. Our results showed that the expression of genes related to the cortisol regulatory process is affected by exposure to high temperature and Cu singly or in combination. The excessive cortisol levels may also be due to the inhibition of cortisol catabolism and excretion, as evidenced by the downregulated expression of Hsd11b2 and Hsd20b2. Hsd11b2 plays a crucial role in converting cortisol into its inactive form cortisone. Hsd20b2, together with Hsd11b2, represents a short pathway in zebra sh to rapidly inactivate and excrete cortisol 10 . Cyp11b1, the key enzyme in cortisol biosynthesis, is also downregulated as a result of Cu toxicity caused by increased temperature.
Signi cant differences in expression have also been observed for Cyp11a1, involving steroid biosynthetic processes in zebra sh exposed to Cu and heat. Cortisol signaling in response to stress is mediated by the GR and mineralocorticoid receptors (MR) genes 30 . In our study, levels of GR mRNA in the zebra sh, activated by cortisol, increased after exposure to Cu and heat stress. The upregulation of GR correlated with the increase in cortisol levels reported during the early developmental stage of the channel cat sh 31 . Furthermore, combined heat and Cu stress signi cantly induced the transcriptional expression of cortisol signaling-related genes, such as Cyp11c1, involved in cortisol metabolic processes; Cyp2k22, involved in oxidation-reduction processes and the cortisol response; Crhb, involved in hormonal regulation of cortisol secretion; Fdx1b, which is an essential mitochondrial redox partner in cortisol biosynthesis; and Foxi3a, which is a key regulator in cortisol synthesis in the adrenal grand. Crhb is elevated in response to environmental stressors such as high temperature in the neuroendocrine system of medaka 32 . Our results indicated that the mRNA levels of Crhb increased on exposure to high temperature and Cu in zebra sh, while there was no signi cant difference in expression in response to Cu exposure alone. The increased expression of Crhb mRNA observed on exposure to heat and Cu is in agreement with results of other stress responses involved in the regulation of cortisol 32,33 . In general, cortisol signaling predominantly occurs through the genomic pathway, involving interaction with GRs and modulating the transcriptional functions of stress-response genes 34 . Cortisol signaling also facilitates rapid responses via non-genomic pathways without gene transcription. The induction of ROS production is suggested as part of the evidence for the action of cortisol through a nongenomic GR-mediated pathway 35 . In the present study, combined exposure to Cu and heat signi cantly upregulated the mRNA levels of antioxidant SOD and GST genes, while the expression of p53 and Bcl-2, which control ROS production and apoptosis in zebra sh, was downregulated.
To conclude, our study is the rst to report that cortisol signaling at the transcriptional level responds to enhanced Cu toxicity caused by increased temperature. Cu is a heavy metal commonly found in the environment. The consideration of an additional factor, such as an increase in temperature, is provided to predict a scenario similar to those caused by global warming. The increase in water temperature affected susceptibility to heavy metals 36,37 . Combined Cu and heat stress may disrupt the signaling pathways involved in cortisol synthesis and catabolism as well as the modulation of intracellular cortisol levels in zebra sh embryo. Finally, the alteration of cortisol signaling might exert negative effects on survival and development in sh by disrupting immune defense systems; these can be used to forecase the effects of global warming.

Materials And Methods
Zebra sh maintenance and exposure conditions. Adult zebra sh used in this study were obtained from the Seoul aquarium (Seoul, Korea) and reared at 26°C with a 14:10 h light-dark cycle in a 3-L acrylic tank using standard methods 38 . Fish were fed TetraMin Flake supplemented with brine shrimp (Artemia salina) thrice daily. Male and female zebra sh (male: female ratio, 1:2) were placed together in spawning tanks (Esen Corp, Beijing) at night. The next morning, eggs were collected within 30 min of spawning and pooled from several spawning tanks. Zebra sh embryos were exposed to CuCl 2 (751944, Sigma-Aldrich, St. Louis, MA, USA) at nominal concentrations of 0.003, 0.007, 0.01, 0.03, 0.07, and 0.1 mg L − 1 at either 26°C or 34°C. At 3-4 hpf, each well of a 12-well plate was lled with 900 µL embryo medium 37 , and 15 embryos were placed in each well. The control group was maintained in only the embryo medium (nontreatment group) at either 26°C or 34°C. All methods for this animal study were approved and carried out in accordance with relevant guidelines and regulations by the Animal Care and Use Committee of Chonnam National University (Yeosu, South Korea). Each experiment was performed in triplicate. Zebra sh tissue samples were stored at − 80°C.
Observation of biological endpoints in zebra sh. After Cu exposure at 26°C or 34°C, survival rates were observed daily until 7 dpf using 15 embryos from each of the treated conditions (six concentrations) and control well plate. The survival rate was determined individually daily. At 35 hpf, the heart rate was observed for 1 min in each group using a Nikon COOLPIX 8700 digital camera (Melville, NY) as described by Park et al., 2020 36,37 . From 2 to 3 dpf, the frequency of malformation was determined by counting zebra sh observed with curved body shape under a stereomicroscope. At 3 dpf, yolk sac edema was determined by measuring the lateral area size of zebra sh larvae anesthetized in 0.03% MS-222 using the ISCapture V3.6 program (TUCSEN Photonics Co. Ltd.) 37 .
Intracellular ROS production and cell death in live zebra sh. In live zebra sh embryos, intracellular ROS production was observed at 3 dpf using a uorescent dye, DCFH-DA or 2,7-dichlorodihydro uorescein diacetate, based on previously established protocols 36 . Cu-induced cell death was detected in zebra sh embryos by staining with acridine orange, a nucleic acid-selective uorescent cationic dye that is used to detect apoptosis 36,37 . Each zebra sh was analyzed using a microscope equipped with a CoolSNAP-Pro color digital camera (Olympus, Japan). The uorescence intensity in the images of each zebra sh larva was determined using the ImageJ software.
Whole-body cortisol measurements. Zebra sh larvae were sacri ced through MS-222 overdose and 10 sh from each group were pooled and immediately frozen on dry ice for cortisol extraction. To extract cortisol, each sample was rinsed and homogenized in 1 ml of 1× PBS. The homogenized samples were stored overnight at − 20°C. After two freeze-thaw cycles were performed to break the cell membranes, the homogenates were centrifuged and the supernatant was removed and assayed immediately. Cortisol levels of the samples were determined using cortisol ELISA kits (CSB-E08487fh, Cusabio, Houston, TX, USA) according to the manufacturer's instructions.
Quantitative real-time PCR (qPCR) and data analysis. The transcription expression of a total of 14 genes were assessed using qPCR. Total RNA was isolated using RNAIsoPlus (Takara, Japan) and treated with recombinant DNase I (Takara, Japan) according to the manufacturers' protocol. The concentration and quality of the RNA was assessed using the Nano-Drop 1000 spectrophotometer (Thermo Fisher Scienti c, Waltham, MA USA) and agarose gel electrophoresis. Complementary DNA was synthesized from 1.5 µg total RNA using the SuperScript™ III RT kit (Invitrogen, Carlsbad, CA, USA). Quantitative PCR was performed on an Exicycler™96 platform (Bioneer, Korea) using AccuPower® GreenStar™ qPCR PreMix (Bioneer, Korea) for SYBR Green-based detection. The PCR conditions used were as follows: 1 cycle at 95°C for 5 min and 40 cycles at 95°C for 10 s and 57-60°C depending on the gene for 40 s. This was followed by a melting curve analysis to ensure single PCR products. The relative level of mRNA expression was calculated using the 2-ΔΔ ct method 39 . The primer information used for qPCR is presented in Supplementary Table 1.
The results are presented as the mean and the standard error of the mean. The mRNA levels of 14 speci c transcripts in each sample were normalized to the combined mean of GAPDH and β-actin 37 . Twoway analysis of variance (ANOVA) was used to analyze signi cant differences between Cu-exposed groups or heat stress and control using SPSS (16.0). Results were considered signi cant at P < 0.05. Figure 1 Biological and developmental responses to different concentrations of Cu, and the non-treated group (control) grown under temperature changes during zebra sh embryogenesis. Survival percentage (%) of zebra sh exposed to six concentrations (0.003, 0.007, 0.01, 0.03, 0.07, and 0.1 mg L−1) at 26 °C (A) and 34 °C (B) up to 7 dpf. Heart rate (C) and yolk sac edema size (%) (D) in zebra sh exposed to different concentrations of Cu at 26 °C and 34 °C. Frequency of malformation (%) in zebra sh exposed to six concentrations of Cu at 26 °C and 34 °C for 3 dpf (E). Differences between exposed and control samples were considered signi cant at P < 0.05.

Figure 2
Cu-induced ROS production and relative expression levels of SOD and GST transcripts in zebra sh embryos at 26 °C (A) and 34 °C (B). ROS levels were measured through image analysis and uorescence microscopy. Embryos were exposed to 0 (control), 0.003, 0.007, and 0.01 mg L−1 Cu. Experiments were performed in triplicate, and the data are presented as mean ± SE. Signi cant effects of Cu concentrations at a given temperature are indicated by * (P < 0.05).

Figure 3
Cu-induced cell death and relative expression levels of p53 and Bcl-2 transcripts in zebra sh embryos at 26 °C (A) and 34 °C (B). Cell death levels were measured using image analysis and uorescence microscopy. Embryos were exposed to 0 (control), 0.003, 0.007, and 0.01 mg L−1 Cu. Experiments were performed in triplicate, and the data are presented as mean ± SE. Signi cant effects of Cu concentrations at a given temperature are indicated by * (P < 0.05).

Figure 4
Cortisol levels in the whole body of zebra sh embryos and heat map based on the relative transcriptional expressions of 14 cortisol signaling and stress-induced genes on zebra sh exposed to different concentrations of Cu (0.003, 0.007, and 0.01 mg L−1), and the non-treated group (control) grown at 26 °C and 34 °C. Experiments were performed in triplicate, and the data are presented as mean ± SE. Signi cant effects of Cu concentrations at a given temperature are indicated by * (P < 0.05).

Supplementary Files
This is a list of supplementary les associated with this preprint. Click to download. 210322SupplTable1Primers.docx