Temperature amplifies cadmium toxicity through bioaccumulation dynamics and hepatic cellular responses in Danio rerio

Temperature amplifies cadmium toxicity through bioaccumulation dynamics and hepatic cellular responses in Danio rerio | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Temperature amplifies cadmium toxicity through bioaccumulation dynamics and hepatic cellular responses in Danio rerio Dola Roy, Madhusmita Mohapatra, Subharthi Pal, Anisa Mitra, Jitendra Kumar Sundaray, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7063175/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 13 You are reading this latest preprint version Abstract Elevated environmental temperatures associated with climate change may potentiate heavy metal toxicity in aquatic ecosystems, yet the mechanisms underlying this interaction remain poorly characterized. This study elucidates how temperature modulates cadmium (Cd) bioaccumulation kinetics and subsequent cellular pathophysiology in adult zebrafish ( Danio rerio ) during chronic exposure (21 days) at control (26°C) versus elevated (34°C) temperatures. Tissue-specific analysis revealed pronounced hepatic Cd accumulation that was significantly amplified (2.4-fold increase) at 34°C compared to 26°C. This temperature-dependent bioaccumulation pattern corresponded with differential metallothionein induction profiles. Histopathological assessment documented progressive hepatocellular deterioration characterized by cytoplasmic vacuolation, sinusoidal dilation, and leukocyte infiltration—effects exacerbated at elevated temperature. Comprehensive biochemical profiling demonstrated marked dysregulation of glucose homeostasis, protein metabolism, lipid parameters, and calcium regulation, with temperature-dependent perturbation patterns. Mechanistic investigations revealed that high temperature synergistically enhanced Cd-induced oxidative stress, evidenced by elevated reactive oxygen species generation, lipid peroxidation, and compensatory antioxidant enzyme modulation. Flow cytometric analysis using Annexin V-FITC/PI and JC-1 staining confirmed that temperature amplified Cd-induced hepatocyte apoptosis through mitochondria-dependent pathways. These findings establish temperature as a critical determinant of Cd toxicokinetics and toxicodynamics in fish, with important implications for ecological risk assessment in thermally fluctuating aquatic environments under climate change scenarios. Bioaccumulation Temperature stress Metal pollution Metallothionein Oxidative stress Apoptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1 Introduction The rapid pace of industrialization and urbanization has resulted in escalating levels of metal pollution in freshwater ecosystems. Human activities such as mining, industrial discharges, and urban runoff are primary contributors to the release of heavy metals into aquatic environments (Sevcikova et al. 2011 ; Castaldo et al. 2021 ). Heavy metal ions are particularly concerning due to their ability to bioaccumulate, lack of biodegradability, and potential toxicity to aquatic organisms (Feng et al. 2015 ). Among these metals, copper (Cu⁺), zinc (Zn²⁺), and cadmium (Cd²⁺) are extensively studied due to their ecological impact and detrimental effects on aquatic life. Cadmium, a non-essential and highly toxic element (McGeer et al. 2011 ), is recognized as a priority pollutant in various countries worldwide (Szebedinszky et al. 2001 ; McGeer et al. 2011 ). Aquatic organisms can accumulate cadmium through direct absorption via gills or ingestion of contaminated food (Perera et al. 2015 ). Once in the body, cadmium is primarily taken up via the divalent metal transporter-1 (DMT1), which is responsible for the absorption of essential metals such as iron and zinc (Bury et al. 2003 ). However, cadmium’s similarity to these essential metals allows it to hijack this transport mechanism, leading to increased accumulation in various tissues. In fish, cadmium uptake through the gills occurs via calcium channels due to its chemical resemblance to calcium ions (Ca²⁺), resulting in competitive inhibition and disruption of calcium homeostasis (McGeer et al. 2011 ; Komjarova and Bury 2014 ). This disruption can interfere with vital physiological functions such as osmoregulation, bone formation, and muscle contraction (Verbost et al. 1989 ; Ramesh et al. 2009 ). Moreover, cadmium can bind to metallothioneins, cysteine-rich proteins that detoxify heavy metals, but excessive exposure leads to the saturation of these binding sites, causing cellular damage and oxidative stress (Samuel et al. 2021 ). Temperature plays a critical role as a physical stressor that regulates survival, growth, and reproduction in ectotherms (Vergauwen et al. 2010 ). Since fish are ectothermic organisms, their body temperature aligns with the surrounding environment, making temperature a crucial factor for their physiological processes. Adaptations to temperature variations occur through physiological modifications and behavioural thermoregulation (Ward et al. 2010 ). Studies on species such as sockeye salmon, zebrafish, and common carp reveal that temperature influences metabolism, osmoregulation, reproduction, and behavior (Crossin et al. 2008 ; Vergauwen et al. 2010 ; Castaldo et al. 2021 ). Elevated temperatures beyond the optimal range can exacerbate the toxic effects of heavy metals and increase metal accumulation due to enhanced metabolic rates (Sokolova and Lannig 2008 ). Prior research has demonstrated that rising temperatures lead to increased cadmium accumulation in Danio rerio (Vergauwen et al. 2013 ; Roy et al. 2024 ) and enhance cadmium elimination rates in stone loach ( Noemacheilus barbatulus ) (Douben 1989 ). Furthermore, studies on Gasterosteus aculeatus exposed to cadmium revealed that temperature elevation consistent with global warming scenarios affected antioxidant parameters, energy reserves, growth patterns, and reproduction (Hani et al. 2018 ). Despite numerous studies on the effects of metals and temperature on fish (Abdel-Tawwab and Wafeek 2017 ; Braz-Mota et al. 2017 ; Li et al. 2021 ), limited information exists on the chronic effects of cadmium exposure and varying temperature scenarios. The present study addresses this gap by examining the chronic toxicity of cadmium in Danio rerio under different temperature stress conditions. To achieve a comprehensive understanding of cadmium toxicity under temperature stress, this study aims to assess cadmium bioaccumulation in Danio rerio tissues, evaluate histopathological alterations in the liver, analyze oxidative stress markers such as reactive oxygen species (ROS) generation and antioxidant enzyme activity, determine cellular apoptosis using Annexin V/PI assay and mitochondrial membrane potential (MMP) disruption, and examine metallothionein expression as a biomarker for cadmium exposure. Moreover, the study seeks to explore the interaction between temperature and cadmium toxicity and its effects on fish physiology. By addressing these objectives, this research aims to provide valuable insights into the ecological risks associated with heavy metal pollution under varying climate conditions. 2 Materials and methods 2.1 Reagents and Chemicals used Analytically pure cadmium chloride (CdCl₂) with a purity of 99.99% was procured from Sigma Aldrich (St. Louis, Missouri, USA) [Product Code: 202908]. A stock solution of CdCl₂ (1 g/L) was prepared by dissolving the appropriate quantity of the salt in deionized water. A standard solution with a concentration of 10 mg/L was prepared using a standard flask. The required concentration of cadmium was achieved by adding a calculated aliquot of the standard solution to the tank using a micropipette. All experimental procedures involving zebrafish were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Calcutta, Kolkata, India. All methods adhered to the applicable guidelines and regulations. 2.2 Fish Management and Experimental Design Adult zebrafish were sourced from a local ornamental fish breeding center in Naihati, West Bengal, India. The experiments were conducted at the Aquatic Bioresource Research Laboratory, Department of Zoology, University of Calcutta, Kolkata, with prior approval from the University’s ethical committee. The fish were bred in the laboratory for two generations before the study. The fish were housed in fiber-reinforced plastic aquaria (2.5 ft x 1 ft x 1 ft) with glass panels on the lateral sides for visibility. The system operated on a flow-through circulatory mechanism, with water filtered through an iron filter near the inlet point. The fish were acclimatized for seven days in dechlorinated tap water, maintained at 25 ± 1°C, pH 7.2 ± 1, salinity 0.3 ± 0.05 mg/L, dissolved oxygen 6.4 ± 1.5 mg/L, ammonia 1 ± 0.5 mg/L, and hardness 215 ± 25 mg/L. A 14:10 light-dark cycle was maintained. The fish were fed live Artemia nauplii once daily and vitamin-enriched flake food twice daily ad libitum. The aquarium water was continuously aerated using stone diffusers connected to an electronic air compressor. One-third of the water was replaced with filtered tap water daily, and the aquaria screens were cleaned regularly. A constant concentration of cadmium chloride was maintained to ensure exposure to the heavy metal. Excess food and fecal matter were removed to maintain water quality. The mortality rate during acclimatization was below 1%. The sub-lethal exposure experiment lasted 21 days, during which fish were exposed to 0.4 mg/L cadmium (1/10th of the 96-hour LC50 at 26°C) at two temperatures: 26°C (optimal) and 34°C (heat shock). Each experimental group consisted of ten fish, with triplicate setups for each condition. A control group was maintained in unchlorinated, aerated tap water. The test solution was replaced every 48 hours, and fish were fed twice daily. At the end of the exposure period, fish were anaesthetized and then euthanized using MS-222 (tricaine methanesulfonate). Whole-body samples were collected for cadmium accumulation analysis, while liver, ovary, gills, muscle, brain, and alimentary canal tissues were dissected and stored at -80°C for further analysis. Fish samples were rinsed with distilled water, and organs were dissected using stainless steel instruments. For whole-body analysis, fish were kept intact after fin removal. 2.3 Rate of bioaccumulation of cadmium in zebrafish Cadmium bioaccumulation in whole fish and various organs (muscle, gill, ovary, brain, and alimentary canal) was evaluated using Atomic Absorption Spectroscopy (AAS), following the APHA 23rd edition (2017). Fish and dissected organs were digested as described by Bawuro et al ( 2018 ). Prior to digestion, all glassware was soaked in 10% nitric acid for 24 hours, rinsed with distilled water, 0.5% (w/v) potassium permanganate solution, and finally distilled water. A 1 mg sample from each organ was digested with a 1:1 mixture of nitric acid and perchloric acid, followed by sulfuric acid. The mixture was heated at 200°C for 30 minutes, cooled, and then made up to 50 ml with distilled water. Cadmium concentration was determined using an Agilent AA55 Atomic Absorption Spectrophotometer, selecting the appropriate wavelengths for analysis. An analytical blank was prepared in the same manner. Results were reported as µg/g wet weight. 2.4 Determination of Metallothionein-2 (MT-2) Concentration, Distribution, and Histopathological Analysis in Fish Tissues Metallothionein-2 (MT-2) concentration and distribution were determined using two methods: Enzyme-Linked Immunosorbent Assay (ELISA) and immunofluorescence. For MT-2 quantification, the ELISA method (Wu et al. 2008 ) was employed using a sandwich kit from Bioassay Technology Laboratory designed for fish MT detection. Whole fish were euthanized, rinsed in PBS, and homogenized on ice. The homogenate was centrifuged, and the supernatant was used for MT-2 quantification. The MT-2 binds to a pre-coated plate with fish MT antibody, followed by biotinylated MT antibody and Streptavidin-HRP. After incubation, unbound Streptavidin-HRP was washed away, and a substrate solution was added to develop color, proportional to MT concentration. The reaction was terminated by adding an acidic stop solution, and absorbance was measured at 450 nm. Results were expressed as ng/ml. For tissue distribution, immunofluorescence was used on cryosectioned tissues. Target organs (gills, muscle, and ovary) were dissected, fixed in paraformaldehyde, cryopreserved in sucrose solutions, and embedded in OCT medium. Sections were then immunolabeled with primary and secondary antibodies, followed by visualization under a fluorescent microscope. The distribution of MT-2 was observed in liver, gill, and ovary tissues. Additionally, histopathological analysis was performed on dissected specimens. Organs were processed by cryosectioning, and tissue sections were stained using the Hematoxylin-Eosin technique for permanent slide preparation. Slides were mounted with DPX, and photomicrographs were taken under an Olympus BX51 microscope to identify any abnormalities in tissue architecture. 2.5 Assessment of histopathological alterations in Cadmium affected zebrafish liver Liver tissues were aseptically dissected on ice and transferred to a Petri dish containing 1X PBS. The samples were processed through Cryosectioning (Campbell et al. 2009 ), with serial sections cut at a thickness of 4–6 µm. The sections were stained using the Hematoxylin-Eosin technique for permanent slide preparation (Deivasigamani 2008 ). Slides were mounted with DPX, and photomicrographs were captured using an Olympus BX 51 compound microscope under appropriate magnification to identify tissue abnormalities. 2.6 Evaluation of biochemical and oxidative stress markers in zebrafish liver under Cadmium exposure 2.6.1 Analysis of biochemical parameters and stress enzyme levels The analysis of biochemical parameters and stress enzyme levels was conducted using specific kits from Precision Biomed Pvt. Ltd, following the methodology of Roy et al. ( 2019 ). All assays were performed with the Robonik Priest Touch Biochemistry Analyser at various filters. Whole fish were euthanized on ice, with fins and heads ablated. The body was cut into small pieces, rinsed in ice-cold phosphate buffer (PBS, pH 7.4) to remove excess blood, and liver tissue was collected for stress enzyme assays. The tissue was homogenized and sonicated at 4°C in PBS to prepare a homogenate, which was centrifuged at 2000–3000 RPM for 20 minutes at 4°C. The supernatant was stored at -80°C until further analysis and thawed at 2–4°C before use. Glucose concentration (mg/dl) was estimated using the Glucose Oxidase-Peroxidase (GOD-POD) method (Trinder 1969 ), with absorbance measured at 500–540 nm. Total protein (g/dl) and direct bilirubin (mg/dl) were assessed via the Biuret method and modified Jendrassik and Grof method (Young 1997 ), both at 546 nm. Cholesterol (mg/dl) was measured with the Cholesterol Oxidase/Peroxidase Aminophenazone i.e., CHOD/PAP method at 510 nm, while calcium (mg/dl) and albumin levels were determined using the Arsenazo III method and BCG method (Young, 1997 ), respectively at 630 nm. Triglycerides were analyzed via the Glycerol Phosphate Oxidase (GPO) method (Young 1997 ) at 510 nm. Alkaline Phosphatase (ALP) activity (U/l) was assessed by converting p-Nitrophenyl phosphate to p-Nitrophenol and phosphate, with absorbance measured at 405 nm every 30 seconds for 90 seconds at 37°C. Aspartate Aminotransferase (AST) activity (mU/ml), or Glutamate Oxaloacetate Transaminase, was measured using the International Federation of Clinical Chemistry method without pyridoxal phosphate with absorbance recorded at 340 nm per minute for 180 seconds (Young, 1997 ). Similarly, Alanine Aminotransferase (ALT) activity (mU/ml), or Glutamate Pyruvate Transaminase, was measured under identical conditions. 2.6.2 Assessment of ROS production in zebrafish liver after Cadmium exposure The intracellular ROS production in the liver of zebrafish, in response to cadmium exposure, was evaluated by assessing phagocytic respiratory burst activity using flow cytometric analysis, following the method described by Pal (2020). Single-cell suspensions were prepared from the liver by treating tissue with Collagenase I (2 mg/ml) at 37ºC with continuous shaking. The cells were then stained for 30 minutes with 2'–7'-Dichlorodihydrofluorescein diacetate (H2-DCFDA), a cell-permeable fluorescent and chemiluminescent probe, in a Ca2+-enriched binding buffer at room temperature in the dark. The ROS generation was measured by analyzing the stained cells using a BD Accuri C6 Flow Cytometer. ROS was detected in the FL1 channel with an emission filter set at 489 nm. The mean fluorescence values of oxidized DCF were recorded and compared between treated and control samples. Data analysis was conducted using BD Accuri C6 software. 2.6.3 Estimation of Oxidative stress enzyme levels in Liver The in-vitro quantitative estimation of oxidative stress enzyme activity, including Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), Glutathione Reductase (GR), Glutathione (GSH), and Lipid Peroxidase (LPO) concentrations in the liver of both control and treated fish was conducted using specific ELISA kits (Bioassay Technology Laboratory, Korain Biotech Co., Ltd.), following the protocol described by Roy et al. ( 2019 ). For sample preparation, liver tissue was aseptically dissected from live, healthy control fish and cadmium- and temperature-treated Danio rerio . The tissue was homogenized with 1 ml of phosphate buffer solution (PBS, pH 7.4) using an automated homogenizer (Remi Motor, India). The homogenate was centrifuged at 2000–3000 RPM for 20 minutes, and the supernatant was collected and stored at -20°C for further analysis. The assay involved adding 50 µl of standard solution and 50 µl of streptavidin-HRP to the standard solution well. For the sample well, 40 µl of the sample was mixed with 10 µl of the respective antibodies (SOD, CAT, GPx, GR, GSH, or LPO) and 50 µl of streptavidin-HRP. The strips were covered with a seal plate membrane, gently shaken to ensure proper mixing, and incubated at 37°C for 60 minutes in the dark. The plate was washed five times with washing buffer, and 50 µl each of Chromogen Solution A and B were added, followed by a 10-minute incubation at 37°C for color development. The reaction was stopped with 50 µl of stop solution, causing a color change from blue to yellow. The absorbance (OD) of each well was measured at 450 nm. The linear regression equation of the standard curve was calculated based on the standards' concentration and corresponding OD values, and the enzyme activity of each sample was determined accordingly. 2.7 Assessment of cell death patterns and mitochondrial membrane potential in zebrafish liver after cadmium exposure The identification of cell death patterns in the liver of Danio rerio was performed using flow cytometry with Annexin V-FITC and Propidium Iodide (PI) dyes, following the methodology described by Pal (2020). The required chemicals included Fluorescein Isothiocyanate (FITC)-conjugated Annexin V, Propidium Iodide, and Annexin binding buffer. Single-cell suspensions were prepared from liver and gill tissues, stained for 30 minutes at room temperature in the dark with FITC-conjugated Annexin V and PI in a calcium-enriched binding buffer, and analyzed using a BD Accuri C6 Flow Cytometer. For each set, a total of 10,000 events were recorded. Annexin V and PI emissions were detected in the FL1 and FL2 channels with emission filters at 508 nm and 643 nm, respectively. The data were analyzed using BD Accuri C6 Software. The mitochondrial membrane potential assay was performed via flow cytometry to evaluate stress-induced apoptotic cell death through mitochondrial depolarization, following Zeng et al ( 2008 ) with minor modifications. JC-1 dye and 1X PBS were used. The cell suspension was centrifuged at 200 g for 5 minutes, and the pellet was resuspended in 1 ml of freshly prepared JC-1 solution. JC-1 accumulates in mitochondria, emitting red fluorescence in cells with intact membrane potential, while apoptotic cells with collapsed membrane potential exhibit green fluorescence (Kumar et al. 2010 ). Both control and treated samples were incubated at 37°C for 15 minutes in the dark and analyzed on a BD Accuri C6 flow cytometer with excitation at 488 nm. Green fluorescence was detected at 530 nm (FL1 channel) and red at 570 nm (FL2 channel), with 10,000 events recorded per set. Data analysis was conducted using BD Accuri C6 Software. 2.8 Statistical analysis Data are presented as mean ± standard error (SE). Statistical analyses were performed using one-way ANOVA followed by Tukey’s multiple comparison test to assess differences between experimental groups. A significance level of p -value < 0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism (Version 7.0, GraphPad Software, USA). 3 Results 3.1 Cadmium Bioaccumulation in Danio rerio The bioaccumulation of cadmium (Cd) was evaluated in whole-body samples (Fig. 1a) and specific tissues, including muscle, gills, liver, alimentary canal, and ovary (Fig. 1b), following 21 days of exposure to 0.4 mg/L Cd at two different temperatures (i.e., 26°C and 34°C). The whole-body Cd accumulation was significantly influenced by temperature, with the highest Cd concentration recorded in the 34°C + Cd group (96.57 ± 2.59 µg/g wet weight). This accumulation was significantly higher ( p -value < 0.0001) compared to both the control group (1.68 ± 0.17 µg/g wet weight, below the measurable threshold) and the 26°C + Cd group (74.24 ± 2.27 µg/g wet weight). Statistical analysis using Tukey’s multiple comparison test further confirmed a significantly higher Cd accumulation ( p -value < 0.0001) in the 26°C + Cd group compared to both untreated groups. However, no significant difference was observed between the control and 34°C untreated groups (1.8 ± 0.18 µg/g wet weight, below the measurable threshold). Organ-specific Cd accumulation, analyzed using Atomic Absorption Spectroscopy (Fig. 1b), revealed that the liver exhibited the highest Cd bioaccumulation, followed by the ovary, gills, muscle, brain, and alimentary canal (Liver > Ovary > Gills > Muscle > Brain > Alimentary Canal). Among the Cd-exposed groups, the highest Cd concentration was detected in the liver of the 34°C + Cd group (32.49 ± 0.38 µg/g wet weight), which was significantly higher ( p -value < 0.0001) than in the ovary (29.29 ± 0.26 µg/g wet weight), gills (17.04 ± 0.22 µg/g wet weight), muscle (11.37 ± 0.16 µg/g wet weight), brain (10.10 ± 0.17 µg/g wet weight), and alimentary canal (6.20 ± 0.20 µg/g wet weight). In contrast, Cd levels in the liver of the control group (0.54 ± 0.09 µg/g wet weight) and the 34°C untreated group (0.76 ± 0.05 µg/g wet weight) remained below threshold limits. However, a significant increase ( p -value < 0.00001) in liver Cd content was observed in the 34°C + Cd group compared to the 26°C + Cd group (28.60 ± 0.18 µg/g wet weight), with a similar trend observed in all other tissues. The Cd concentrations in different organs are summarized in Supp. Table 1 . Tukey’s multiple comparison test further revealed a significant difference ( p -value < 0.001) in Cd bioaccumulation between the 26°C + Cd and 34°C + Cd groups across all analyzed tissues. Table 1 Semi-quantitative scoring of histopathological alterations in the liver of Danio rerio after 21 days of exposure to 0.4 mg/l Cd at two temperatures (26°C and 34°C). The severity of lesions is graded as follows: (-) none, (+) mild, (++) moderate, and (+++) severe. Condition 26° Control 34° Control 26° Treated 34° Treated 1. Blood congestion in sinusoid - + ++ +++ 2. Swelling of hepatocyte - - ++ ++ 3. Presence of dark granules - - +++ +++ 4. Cellular degeneration - - +++ ++ 3.2 Metallothionein concentration as a biomarker for cadmium-induced stress in Danio rerio The concentration of cellular stress protein, Metallothionein is a good indicator of water pollution and also reported as a good bioindicator of heavy metals pollution. Metallothioneins (MT) are cysteine rich proteins able to sequester Cd ions. Since MT is found in several organs like liver, kidney, muscle etc., we have determined the concentration in whole organism (Fig. 2a). The results indicated that, MT concentration increases synchronously with Cd accumulation rates and likewise the highest level was found in 34°C + Cd group of fish. Dunnett’s Multiple comparison test revealed that, there is a significant difference ( p -value < 0.0001) between control group (16.02 ± 1.09 ng/ml) and 34°C treated group (25.60 ± 1.19 ng/ml) which indicates that temperature has a potential effect on MT concentration. The Cd treated groups both at 26°C (67.60 ± 0.96 ng/ml) and 34°C (67.06 ± 1.9 ng/ml) showed significant difference ( p -value < 0.0001). 3.2.1 Immunofluorescence localization of metallothionein in liver of Danio rerio Immunopositivity reactions for Metallothionein (MT) was determined in the cells of liver (Fig. 2b) for all four experimental groups. Immunofluorescence analysis revealed substantial localization of MT in the tissues of Cd treated groups. The immunopositivity for MT protein showed an increase in groups treated with Cd at both 26°C and 34°C but the untreated groups showed basal level of MT expression. While at the most intense MT protein expression was observed in group treated with both Cd and high temperature (34°C). The immunodetection for MTs shows the absence of signal in the tissues of the animals from the control group. The liver tissues showed a marked increase in the Immunoreactivity of MTs in the Cd treated group at 34°C compared to the one at optimum temperature (26°C). MT was found to be distributed both in the cytoplasmic and nuclear compartments of the hepatocytes (Fig. 2b). Since, the hepatocytes are tightly packed, strong immunopositivity is found among the Cd treated groups (Fig. 2b). No immunofluorescence was found in the untreated groups of fishes. 3.3 Histopathological analysis of cadmium-induced alterations in Danio rerio liver tissue A light microscopic analysis of cadmium affected Danio rerio liver tissue revealed distinct structural features across experimental groups (Fig. 3). The control group (26°C) exhibited a normal liver architecture with polyhedral hepatocytes (HS), centrally located nuclei, and prominent nucleoli (Fig. 3A). Blood sinusoids (BS), lined with reticulo-endothelial cells, were surrounded by hepatocytes, while bile canaliculi were found near the portal vein, lined with simple cuboidal epithelium. Lipid glycogen granules were also present in the cytoplasm (Fig. 3A). Exposure to elevated temperature (34°C) resulted in mild blood congestion in the sinusoids and hydropic swelling of hepatocytes, though the nuclei retained a nearly normal shape. A substantial number of normal hepatocytes were observed in this group (Fig. 3B). Cd exposure at both optimum (26°C) and high (34°C) temperatures led to the accumulation of dark granules in some hepatocytes (Fig. 3C and D). Histopathological alterations included degenerated hepatic tissue, vacuole formation, blood cell infiltration, cellular swelling, nuclear degeneration, karyorrhexis, loss of hepatocytic cell walls, and disorganized hepatic cords. The liver morphology became fragmented, with no compact structure as seen in the control group. The 34°C + Cd group exhibited numerous lipid droplets, dark granule accumulation, loss of hepatocyte polyhedral architecture, and lobular disruption. Fat degeneration resulted in irregular clear spaces between hepatocytes, along with mild hypertrophy and widened sinusoids (Fig. 3D). A Semi-Quantitative Scoring was performed to assess the relative severity of histopathological lesions, as shown in Table 1 . 3.4 Biochemical alterations in liver tissue after cadmium exposure and elevated temperature in Danio rerio Changes in the biochemical parameters of Control, 34°C treated and Cd treated at 26°C and 34°C are summarized in Fig. 4. After 21 days of chronic Cd exposure significant changes were found in the liver tissue for all parameters i.e. Glucose, Total protein, Cholesterol, Bilirubin, Triglyceride and Calcium. Tukey’s multiple comparison test revealed that, glucose level had increased significantly ( p -value < 0.001, df = 3) in groups exposed to Cd as well as a combination of higher temperature with cadmium compared to control fishes. Whereas, no significant difference was found among the 26°C and 34°C untreated groups (Fig. 4a). The highest value of glucose was found in the 34°C + Cd treated group (224.4 ± 8.75 mg/dl) followed by 26°C + Cd treated group (63.24 ± 1.99 mg/dl), 34°C group (41.60 ± 0.21 mg/dl) and Control fishes (27.61 ± 1.54 mg/dl). A significant reduction in Total Protein, Cholesterol, and Triglyceride levels was observed in cadmium-exposed groups compared to the control (Fig. 4b, 4c, 4d). Tukey’s multiple comparison test showed a notable decline in Total Protein levels ( p -value < 0.001, df = 3), with the lowest value in the 34°C + Cd group (0.644 ± 0.018 g/dl), followed by the 26°C + Cd group (0.735 ± 0.021 g/dl). Cholesterol levels significantly dropped in Cd-treated groups with the lowest in the 26°C + Cd group (9.81 ± 0.13 mg/dl), followed by 34°C + Cd (13.98 ± 0.29 mg/dl). Triglyceride levels also decreased significantly, with the 26°C + Cd group recording the lowest (47.13 ± 0.66 mg/dl), which was not significantly different from the 34°C + Cd group (49.0 ± 0.73 mg/dl). Bilirubin levels significantly increased in all Cd-treated groups, with the highest concentration in the 34°C + Cd group (7.11 ± 0.22 mg/dl) (Fig. 4e). Calcium levels decreased significantly across all treated groups, with the 26°C + Cd group showing the lowest (1.67 ± 0.069 mg/dl) (Fig. 4f). Correlation analysis based on Pearson’s correlation coefficient ( p -value < 0.05) was performed among biochemical parameters with an aim to find linear relation between analyzed parameters. Further, heatmap was used to visualize hierarchical clustering among the biochemical parameters (using GraphPad Prism 8.0.1). According to Pearson’s correlation coefficient, a strong positive correlation was found between Glucose and Bilirubin content ( r = 0.870) (Supp. Figure 1). Total protein was positively correlated with Cholesterol ( r = 0.826), Triglyceride ( r = 0.930) and Calcium content ( r = 0.703). Similarly, Cholesterol content was found to be positively correlated with Triglyceride ( r = 0.880) and Calcium ( r = 0.917). Although a strongly negative correlation was found between Total protein and Bilirubin ( r = -0.877); Bilirubin and Triglyceride ( r = -0.819). 3.4.1 Oxidative stress response through ROS in Liver Cadmium exposure significantly ( p -value < 0.0001) increased ROS production in all treated groups compared to the control (Supp. Figure 2). The highest ROS level was observed in the 34°C + Cd group (62950 ± 86.13), significantly higher than the 26°C + Cd group (48704 ± 163.9). Notably, the 34°C Cd-untreated group (38671 ± 204.1) also exhibited elevated ROS levels compared to the control (25666 ± 107.6), indicating temperature-induced oxidative stress. 3.4.2 Evaluation of antioxidant and stress enzyme activities in liver SOD and CAT activities were significantly elevated in Cd-exposed groups ( p -value < 0.0001; Fig. 5a, 5b), with the highest levels observed in the 34°C + Cd group (SOD: 158.9 ± 1.02 U/mg Protein/min; CAT: 43.0 ± 0.37 U/mg Protein/min). No significant difference was found between the 34°C Cd-untreated and control groups for CAT activity (Fig. 5b). Furthermore, GSH , GPx , and GR activities were also significantly increased in Cd-treated groups ( p -value < 0.0001; Fig. 5c-e). The 26°C + Cd group exhibited the highest enzyme activities ( GSH : 12.24 ± 0.29 nmole/mg Protein/min; GPx: 21.72 ± 0.36 nmole/mg Protein/min; GR: 16.45 ± 0.28 nmole/mg Protein/min), significantly higher than the 34°C + Cd group and controls. Tukey’s test indicated no significant difference in GPx activity between the 34°C Cd-untreated and control groups, though GSH and GR levels were significantly different. Cadmium exposure significantly increased ALP, AST, and ALT concentrations ( p -value < 0.0001; Fig. 5f-h). The 34°C + Cd group showed the highest ALP levels (8.89 ± 0.16 U/L), significantly greater than the 26°C + Cd group (6.82 ± 0.23 U/L) and controls. AST and ALT levels followed a similar trend, with significant increases in treated groups at both temperatures compared to controls. LPO levels increased substantially in Cd-treated groups (Fig. 5i), with the highest concentration in the 34°C + Cd group (56.64 ± 0.46 nmole/ml), significantly exceeding the 26°C + Cd group (41.60 ± 0.32 nmole/ml) and controls. Notably, LPO levels in the 34°C untreated group were also significantly ( p -value < 0.001) higher than controls According to the correlation analysis based on Pearson’s correlation coefficient a very strong positive correlation was found among the antioxidant enzymes with the stress enzymes. SOD has strong positive correlation with all the stress parameters except GPx ( r = 0.736), similarly CAT shown very low positive correlation with GPx ( r = 0.360) as well. A very strong positive correlation was found between GSH, GPx and GR whereas LPO was found to be more positively correlated with ALP, AST and ALT ( r = 0.994, r = 0.971 and r = 0.992 respectively). 3.5 Apoptotic Analysis in Liver Tissue of Danio rerio: Annexin V-FITC/PI Staining and MMP Assay Annexin V-FITC/PI staining analysis demonstrated apoptosis induction in the liver of Danio rerio across four experimental groups (Fig. 6a; Supp. Figure 3a-d). Double-negative staining indicates live cells (LL), Annexin V-FITC positive and PI negative staining represents early apoptosis (LR), and double-positive staining indicates late apoptosis (UR) (Supp. Figure 3a-d). Tukey’s Multiple Comparison Test revealed a significant increase ( p -value < 0.001) in both early and late apoptotic cells in Cd-treated groups compared to the control. The percentage of normal cells significantly declined in treated groups (Fig. 6a). The highest percentage of early apoptotic cells was observed in the 26°C + Cd group (18.27 ± 0.38), followed by the 34°C + Cd group (15.08 ± 0.31), 34°C untreated group (10.56 ± 0.37), and control group (6.20 ± 0.37), with a significant difference ( p -value < 0.0001) between treated and control groups. For late apoptotic cells, a significant increase was observed in the 26°C + Cd (1.10 ± 0.10) and 34°C + Cd (4.80 ± 0.21) groups compared to the control (0.29 ± 0.03), while no significant difference was found between the 34°C untreated group (0.27 ± 0.02) and the control. The MMP assay in the liver of Danio rerio (Fig. 6b; Supp. Figure 3e-h) revealed that the disruption of active mitochondria, a hallmark of early apoptosis, leads to changes in membrane potential. In healthy cells, JC-1 accumulates in mitochondria, forming J-aggregates with red fluorescence. However, in apoptotic cells with reduced MMP, JC-1 remains in monomeric form, emitting green fluorescence. In the control group, most cells maintained normal MMP (ΔΨm), showing bright fluorescence in both BL1-A and BL3-A channels (BL1-A bright, BL3-A bright) (Supp. Figure 3e and f). In contrast, cells in both temperature- and Cd-treated groups exhibited reduced BL3-A intensity (BL1-A bright, BL3-A dim), indicating compromised ΔΨm (Supp. Figure 3g and h). The collapse of ΔΨm is associated with mitochondrial depolarization, which can trigger cytochrome c release and apoptosis. The percentage of polarized cells significantly decreased ( p -value < 0.001) in the treatment groups, with the lowest percentage observed in the 34°C + Cd group (Fig. 6b). Additionally, an increase in depolarized cells in the 34°C untreated group suggests that elevated temperature alone can induce cell death. The percentage of cells with polarized MMP was significantly lower in the Cd-treated groups (26°C, 74.26 ± 0.39; 34°C, 68.64 ± 0.77) compared to the control (86.25 ± 0.27) and 34°C untreated group (80.47 ± 0.42). The highest percentage of depolarized cells was found in the 34°C + Cd group (31.37 ± 0.76), followed by the 26°C + Cd group (25.74 ± 0.39), 34°C untreated group (19.53 ± 0.42), and control (13.75 ± 0.27). 4 Discussion Cd bioaccumulation in aquatic environments poses a significant risk to human health through fish consumption. Key factors influencing Cd accumulation in fish tissues include environmental concentration, exposure duration, temperature, and pH. This study demonstrated a tissue-specific accumulation pattern, with the liver exhibiting the highest concentration (Fig. 1b) due to its role in detoxification and metabolic functions. The order of accumulation was Liver > Ovary > Gills > Muscles > Brain > Alimentary canal. Elevated temperatures further amplified Cd accumulation, especially in the liver and ovaries. The gills, serving as the primary entry point for Cd due to their extensive surface area and vascular structure, also showed substantial accumulation (Fig. 1b). In contrast, the brain and muscles had lower Cd levels due to the protective blood-brain barrier and limited detoxification activity. The study highlights that both Cd exposure and elevated temperatures significantly influence bioaccumulation and toxicity in Danio rerio . Waterborne Cd can cause oxidative stress, immunosuppression, and endocrine disruption, ultimately affecting growth and survival (Cao et al. 2010 ; Kumar and Singh 2010 ; Wen et al. 2018 ). The observed temperature-dependent increase in Cd accumulation, particularly in the liver, underscores the synergistic effect of elevated temperature on metal retention. These findings align with previous research showing that higher temperatures enhance the uptake and retention of toxic metals in aquatic organisms (Wen et al. 2018 ; Moiseenko et al. 2020; Castaldo et al. 2021 ; Yang et al. 2022 ). MT is known to sequester heavy metals, thereby protecting cells from toxicity, but its elevated levels also reflect the burden of Cd exposure in the organism (Ruttkay-Nedecky et al. 2013 ). This study confirms that exposure to metals usually promotes the induction of MT (Fig. 2a), which is consistent with the previous studies (Shariati et al. 2011 ; Wen et al. 2018 ). In addition to increased Cd bioaccumulation, co-exposure to Cd resulted in an elevated MT concentration in D. rerio (Lu et al. 2018 ). Such distinct results indicate that MT induction is positively correlated with the accumulation of Cd in fish tissue. The significant correlation between Cd levels and MT concentrations further supports the role of MT as a reliable biomarker for Cd exposure and toxicity. Histological analysis serves as a valuable biomarker for assessing the impact of xenobiotic compounds on biological systems (Paolini et al. 2005 ). In this study, cadmium-induced histopathological alterations in the liver, including cellular swelling, vacuole formation, and disrupted hepatic architecture, align with previous findings on cadmium's hepatotoxicity in fish (Chavan and Muley 2014 ; Kaur et al. 2018 ). Severe lesions such as lymphonuclear cell infiltration, degeneration of hepatic parenchyma, and hepatocyte deformation were observed in L. rohita exposed to heavy metals (Kaur et al. 2018 ). Similar pathological changes, including cytoplasmic vacuolation and focal necrosis, were reported in C. mrigala exposed to lead acetate (Chavan and Muley 2014 ). Additionally, Tilapia mossambica exhibited severe damage and vacuolation after exposure to cadmium sulfate (Jalaludeen et al. 2012 ). Cadmium exposure also caused hypertrophy, vacuolation, and necrosis in Cyprinus carpio (Patnaik et al. 2011 ), while C. mrigala showed complete disintegration of liver tissue when exposed to lead nitrate (Mary et al. 2014 ). These findings highlight the liver’s role as a primary detoxification site and the accumulation of cadmium bound to metallothionein, leading to prolonged histopathological damage (Olsson and Hogstrand 1987 ). Such structural changes serve as crucial indicators of heavy metal toxicity in aquatic environments. The rise in glucose levels in Danio rerio exposed to Cd and high temperatures (Fig. 4a) indicates metabolic disruption, likely due to impaired liver function. This increase stems from gluconeogenesis, which supplies energy for detoxification (Zutshi et al. 2010 ; Kavitha et al. 2010 ), and is driven by stress hormones like cortisol and catecholamines that trigger glycogenolysis (Randall and Ferry 1992 ). Similar patterns were observed in Mugil cephalus (Hilmy et al. 1987 ), while Clarias batrachus exhibited reduced glucose levels when exposed to Cd and Hg (Arya 2014 ). The findings suggest that Cd-induced stress, amplified by elevated temperatures, elevates glucose as an adaptive energy response. The observed reduction in protein content in Cd-exposed fish (Fig. 4b) may result from impaired protein synthesis or increased protein breakdown due to metabolic stress, possibly from nephrosis or cirrhosis (Arya 2014 ). Excessive proteolysis is likely occurring to meet energy demands during Cd exposure, as reflected by elevated glucose levels. Similar protein reductions have been reported in various fish species exposed to heavy metals (Cicik and Engin 2005 ; Binukumari et al. 2016 ). Additionally, increased protease activity and proteolysis contribute to energy production during Cd stress. Unconjugated bilirubin, a product of heme catabolism, showed a significant increase in Cd-exposed groups, particularly at higher temperatures. This elevation could indicate hemolysis or impaired bilirubin conjugation. Elevated bilirubin levels are commonly associated with stress responses (Levitt and Levitt 2014 ), although conjugation in zebrafish is not well studied (Liu et al. 2013 ). A decrease in cholesterol levels was observed in the Cd-exposed fish (Fig. 4c), possibly due to kidney tissue damage and membrane disruption. Conversely, an increase in cholesterol was reported in O. niloticus (Öner et al. 2008 ). The reduction in cholesterol suggests environmental stress and increased lipid utilization to meet energy demands. Similarly, a decline in triglycerides was noted, likely due to impaired lipid metabolism and glycogen storage (Heydarnejad et al. 2013 ). Calcium levels were significantly lower in the Cd-exposed groups (4f), with the greatest reduction at elevated temperatures. Similar declines were observed in other species exposed to Cd (Honda and Suzuki 2020 ). Reduced calcium may disrupt vitellogenin (VTG) transport and ovarian maturation in D. rerio , as calcium has a positive correlation with VTG, particularly during yolk deposition (Linares-Casenave et al. 2003 ). The correlation matrix reveals a significant negative correlation between glucose and total protein (Supp. Figure 1), consistent with Gagnon et al. ( 2006 ), who observed similar trends in fish under metal stress. This suggests that elevated glucose levels may be linked to liver damage and renal dysfunction (Pratap and Wendelaar Bonga 1990 ), as supported by the positive correlation between glucose and bilirubin. A study on Cyprinus carpio by Cicik and Engin ( 2005 ) showed similar interactions. Additionally, total protein levels were positively correlated with triglycerides and cholesterol, indicating liver dysfunction, reduced absorption, and protein loss (Heydarnejad et al. 2013 ), but showed a negative correlation with bilirubin. The positive correlation between calcium and cholesterol aligns with Linares-Casenave et al ( 2003 ), highlighting the role of cholesterol synthesis in VTG production. Oxidative stress, as evidenced by increased ROS levels and enhanced antioxidant enzyme activities, is a critical mechanism underlying cadmium toxicity. The 34°C + Cd group exhibited the highest ROS production, indicating that elevated temperature exacerbates oxidative damage. Similar findings have been reported in other studies, where temperature increases led to enhanced oxidative stress in fish exposed to pollutants (Vergauwen et al. 2013 ). The SOD-CAT system is usually regarded as the first line of defense towards the production of ROS under environmental stressors (Pandey et al. 2003 ). The superoxide radical can be turned into H 2 O 2 by SOD, while H 2 O 2 is metabolized by CAT. In this study, Cd led to CAT activation in zebrafish (Fig. 5b), probably due to the antioxidative response to elevated H 2 O 2 production (Lu et al. 2018 ). GSH, a vital non-enzymatic antioxidant, plays a crucial role in fish immune functions. It reacts with ROS to form glutathione disulfide and serves as a substrate in detoxifying exotic toxic chemicals through biotransformation catalyzed by glutathione S-transferase (Zhang et al. 2013 ). Glutathione reductase (GR) is responsible for maintaining cellular redox balance by regulating the GSH/GSSG ratio (Sarkar et al. 2014 ). The observed changes in antioxidant enzyme activities, such as increased SOD, CAT, and GSH activities, reflect the organism's response to mitigate oxidative damage (Company et al. 2004 ; Banni et al. 2011 ). However, the reduced efficiency of the antioxidant defence system at higher temperatures suggests an overwhelmed protective response under these conditions. Cd-induced apoptosis, a critical response to cellular damage, was confirmed through Annexin V-FITC/PI staining and mitochondrial membrane potential (MMP) assays. The study revealed a significant increase in early and late apoptotic cells, especially in the 34°C + Cd group (Fig. 6a), indicating that elevated temperature enhances cadmium-induced cell death via mitochondrial dysfunction. This aligns with previous findings on cadmium’s ability to disrupt MMP and trigger apoptosis (Zhang et al. 2019 ). Cadmium exposure leads to oxidative stress, DNA damage, and mitochondrial dysfunction, which activates the intrinsic apoptotic pathway through the release of cytochrome c and caspase activation (Jiang and Wang 2004 ; Hosseini et al. 2014 ). The elevated production of ROS impairs mitochondrial membrane permeability, resulting in apoptosis. The current study demonstrated that cadmium exposure significantly increased apoptotic hepatocytes and MMP depolarization in D. rerio , with higher apoptotic rates observed at elevated temperatures. These findings are consistent with prior research on heavy metal-induced apoptosis in fish and other organisms (Gao et al. 2013 ; Liu et al. 2020 ). The results highlight the critical role of mitochondrial dysfunction in cadmium-induced hepatic apoptosis and the amplifying effect of temperature on this process. 5 Conclusion This study provides compelling evidence that elevated temperature significantly exacerbates the toxic effects of cadmium in Danio rerio , leading to increased bioaccumulation across various organs, including muscle, gill, ovary, brain, and alimentary canal. The atomic absorption spectroscopy analysis revealed a substantial rise in cadmium concentration, particularly in metabolically active tissues like the liver. Elevated temperature not only intensified cadmium accumulation but also triggered heightened oxidative stress, as indicated by increased levels of reactive oxygen species and antioxidant enzyme activity. Histopathological examination showed severe tissue damage, including cellular degeneration, necrosis, and vacuolization, while apoptotic markers confirmed enhanced cell death in liver tissue. These findings underscore the synergistic impact of temperature and cadmium exposure, highlighting the vulnerability of aquatic organisms to heavy metal toxicity in the context of climate change. Moreover, this study emphasizes the need for future research to elucidate the molecular pathways involved in temperature-mediated metal toxicity and to develop effective mitigation strategies to safeguard aquatic ecosystems from the dual threats of global warming and heavy metal contamination. Declarations Acknowledgements The authors are thankful to the Head of the Department of Zoology, University of Calcutta, India for providing the facilities. The authors are also thankful to the Director, Zoological Survey of India. Funding This study was funded by Council of Scientific & Industrial Research, Senior Research Fellowship (Direct) Programme, Government of India [Sanction No. 09/028(1036)/2018-EMR-I Dated: 16.04.2018]. The authors are also grateful to DST SERB NPDF scheme [Sanction No. PDF/2023/000069] for funding. Authors’ Contributions DR and MM: Conceptualization, data curation, methodology, writing - original draft and editing); SP, AM and JKS: review and editing and SHC (conceptualization, supervision, project administration, editing). Competing Interests The authors declare no competing interests that could have appeared to influence the work reported. Data Availability Statement The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request. Ethical Approval All experimental procedures involving zebrafish were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Calcutta, Kolkata, India. All methods adhered to the applicable guidelines and regulations. Consent to Participate Not applicable. Consent to Publish I, Roy, D., the corresponding author of this article hereby confirms that all coauthors have agreed on the final version of this manuscript to be published according to the journal guidelines. References Abdel-Tawwab M, Wafeek M (2017) Fluctuations in water temperature affected waterborne cadmium toxicity: hematology, anaerobic glucose pathway, and oxidative stress status of Nile tilapia, Oreochromis niloticus (L.). Aquac 477: 106-111. 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Additional Declarations No competing interests reported. Supplementary Files SuppFigure1.jpg SuppFigure2.jpg SuppFigure3.jpg Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 05 Sep, 2025 Reviews received at journal 31 Aug, 2025 Reviews received at journal 28 Aug, 2025 Reviews received at journal 27 Aug, 2025 Reviewers agreed at journal 20 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers invited by journal 11 Aug, 2025 Editor assigned by journal 08 Aug, 2025 Editor invited by journal 06 Aug, 2025 Submission checks completed at journal 25 Jul, 2025 First submitted to journal 25 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Roy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA30lEQVRIiWNgGAWjYDCCAyCiQkJOnr0ByDCwIFbLGQtjwx4Qy0CCSC2MbRWJDDcSQFwitPAd4H34mLdNIoFx5vOrG34USDDwt3cn4NUieYDd2JjnnEQeu3RO2c0eoMMkzpzdgFeLwQE2NmmeMolixtk5aTd4gFoMJHKJ0cImkdhw80zazT/Ea2kDarnBfuw2UbZIHmZjNpxzRgIYyDlst2UMJHgI+oXveBvjgzcVdcCoPP7s5ps/NnL87b34tTAwMzAw8YBZPAZgEr9yKGD8AabYHxClehSMglEwCkYeAADKH0SZtRHG3wAAAABJRU5ErkJggg==","orcid":"","institution":"Zoological Survey of India","correspondingAuthor":true,"prefix":"","firstName":"Dola","middleName":"","lastName":"Roy","suffix":""},{"id":501728671,"identity":"9aba69bf-026d-467f-bfcf-0fb0466e5f8f","order_by":1,"name":"Madhusmita Mohapatra","email":"","orcid":"","institution":"ICAR- Central Institute of Freshwater Aquaculture","correspondingAuthor":false,"prefix":"","firstName":"Madhusmita","middleName":"","lastName":"Mohapatra","suffix":""},{"id":501728672,"identity":"dea4c03a-fa90-4ac4-838d-1061d9e41eb3","order_by":2,"name":"Subharthi Pal","email":"","orcid":"","institution":"Bhairab Ganguly College","correspondingAuthor":false,"prefix":"","firstName":"Subharthi","middleName":"","lastName":"Pal","suffix":""},{"id":501728673,"identity":"719c75b5-b30f-4d5c-baa9-efb910bb2339","order_by":3,"name":"Anisa Mitra","email":"","orcid":"","institution":"Sundarban Hazi Desarat College","correspondingAuthor":false,"prefix":"","firstName":"Anisa","middleName":"","lastName":"Mitra","suffix":""},{"id":501728674,"identity":"00563368-0f6c-448e-b385-13c879dc971b","order_by":4,"name":"Jitendra Kumar Sundaray","email":"","orcid":"","institution":"ICAR- Central Institute of Freshwater Aquaculture","correspondingAuthor":false,"prefix":"","firstName":"Jitendra","middleName":"Kumar","lastName":"Sundaray","suffix":""},{"id":501728675,"identity":"4d8b7684-3ce1-483c-a3e1-dc7e2350ee79","order_by":5,"name":"Sumit Homechaudhuri","email":"","orcid":"","institution":"University of Calcutta","correspondingAuthor":false,"prefix":"","firstName":"Sumit","middleName":"","lastName":"Homechaudhuri","suffix":""}],"badges":[],"createdAt":"2025-07-07 08:38:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7063175/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7063175/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89399777,"identity":"b228d39d-8a54-4002-a0c6-c5981912c90c","added_by":"auto","created_at":"2025-08-19 14:07:10","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":105712,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure 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legend.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7063175/v1/4b298daff67f59184876aff3.jpg"},{"id":89402390,"identity":"0fd2fcd4-d4bc-422b-9150-9c529bee65cd","added_by":"auto","created_at":"2025-08-19 14:31:10","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":130565,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7063175/v1/28566e791e71fcfd3ec65925.jpg"},{"id":89402389,"identity":"976111bd-fbd3-4a27-a780-1b10b020f1b2","added_by":"auto","created_at":"2025-08-19 14:31:10","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":184328,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure 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14:39:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2198280,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7063175/v1/1481d850-54de-4a9c-8497-5b087e6a779d.pdf"},{"id":89400724,"identity":"f30f97a5-e8c2-4323-b3a0-a30242de8c2b","added_by":"auto","created_at":"2025-08-19 14:15:10","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":93600,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7063175/v1/aaf6fc2febe04e67fab4db1c.jpg"},{"id":89399780,"identity":"6c868913-59ef-4456-badc-62b2a11831be","added_by":"auto","created_at":"2025-08-19 14:07:10","extension":"jpg","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":104271,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7063175/v1/32076f97ad17c98c32bd2b0e.jpg"},{"id":89399786,"identity":"528dcc16-2ba3-4edb-86ca-1ae3a79fb2a4","added_by":"auto","created_at":"2025-08-19 14:07:10","extension":"jpg","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":163421,"visible":true,"origin":"","legend":"","description":"","filename":"SuppFigure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7063175/v1/b37c35fca9f99217d4d601a0.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Temperature amplifies cadmium toxicity through bioaccumulation dynamics and hepatic cellular responses in Danio rerio","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eThe rapid pace of industrialization and urbanization has resulted in escalating levels of metal pollution in freshwater ecosystems. Human activities such as mining, industrial discharges, and urban runoff are primary contributors to the release of heavy metals into aquatic environments (Sevcikova et al. \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Castaldo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Heavy metal ions are particularly concerning due to their ability to bioaccumulate, lack of biodegradability, and potential toxicity to aquatic organisms (Feng et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Among these metals, copper (Cu⁺), zinc (Zn\u0026sup2;⁺), and cadmium (Cd\u0026sup2;⁺) are extensively studied due to their ecological impact and detrimental effects on aquatic life. Cadmium, a non-essential and highly toxic element (McGeer et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), is recognized as a priority pollutant in various countries worldwide (Szebedinszky et al. \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; McGeer et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAquatic organisms can accumulate cadmium through direct absorption via gills or ingestion of contaminated food (Perera et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Once in the body, cadmium is primarily taken up via the divalent metal transporter-1 (DMT1), which is responsible for the absorption of essential metals such as iron and zinc (Bury et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, cadmium\u0026rsquo;s similarity to these essential metals allows it to hijack this transport mechanism, leading to increased accumulation in various tissues. In fish, cadmium uptake through the gills occurs via calcium channels due to its chemical resemblance to calcium ions (Ca\u0026sup2;⁺), resulting in competitive inhibition and disruption of calcium homeostasis (McGeer et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Komjarova and Bury \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This disruption can interfere with vital physiological functions such as osmoregulation, bone formation, and muscle contraction (Verbost et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e1989\u003c/span\u003e; Ramesh et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Moreover, cadmium can bind to metallothioneins, cysteine-rich proteins that detoxify heavy metals, but excessive exposure leads to the saturation of these binding sites, causing cellular damage and oxidative stress (Samuel et al. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTemperature plays a critical role as a physical stressor that regulates survival, growth, and reproduction in ectotherms (Vergauwen et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Since fish are ectothermic organisms, their body temperature aligns with the surrounding environment, making temperature a crucial factor for their physiological processes. Adaptations to temperature variations occur through physiological modifications and behavioural thermoregulation (Ward et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Studies on species such as sockeye salmon, zebrafish, and common carp reveal that temperature influences metabolism, osmoregulation, reproduction, and behavior (Crossin et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Vergauwen et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Castaldo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Elevated temperatures beyond the optimal range can exacerbate the toxic effects of heavy metals and increase metal accumulation due to enhanced metabolic rates (Sokolova and Lannig \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Prior research has demonstrated that rising temperatures lead to increased cadmium accumulation in \u003cem\u003eDanio rerio\u003c/em\u003e (Vergauwen et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Roy et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) and enhance cadmium elimination rates in stone loach (\u003cem\u003eNoemacheilus barbatulus\u003c/em\u003e) (Douben \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1989\u003c/span\u003e). Furthermore, studies on \u003cem\u003eGasterosteus aculeatus\u003c/em\u003e exposed to cadmium revealed that temperature elevation consistent with global warming scenarios affected antioxidant parameters, energy reserves, growth patterns, and reproduction (Hani et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eDespite numerous studies on the effects of metals and temperature on fish (Abdel-Tawwab and Wafeek \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Braz-Mota et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), limited information exists on the chronic effects of cadmium exposure and varying temperature scenarios. The present study addresses this gap by examining the chronic toxicity of cadmium in \u003cem\u003eDanio rerio\u003c/em\u003e under different temperature stress conditions. To achieve a comprehensive understanding of cadmium toxicity under temperature stress, this study aims to assess cadmium bioaccumulation in \u003cem\u003eDanio rerio\u003c/em\u003e tissues, evaluate histopathological alterations in the liver, analyze oxidative stress markers such as reactive oxygen species (ROS) generation and antioxidant enzyme activity, determine cellular apoptosis using Annexin V/PI assay and mitochondrial membrane potential (MMP) disruption, and examine metallothionein expression as a biomarker for cadmium exposure. Moreover, the study seeks to explore the interaction between temperature and cadmium toxicity and its effects on fish physiology. By addressing these objectives, this research aims to provide valuable insights into the ecological risks associated with heavy metal pollution under varying climate conditions.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Reagents and Chemicals used\u003c/h2\u003e\u003cp\u003eAnalytically pure cadmium chloride (CdCl₂) with a purity of 99.99% was procured from Sigma Aldrich (St. Louis, Missouri, USA) [Product Code: 202908]. A stock solution of CdCl₂ (1 g/L) was prepared by dissolving the appropriate quantity of the salt in deionized water. A standard solution with a concentration of 10 mg/L was prepared using a standard flask. The required concentration of cadmium was achieved by adding a calculated aliquot of the standard solution to the tank using a micropipette. All experimental procedures involving zebrafish were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Calcutta, Kolkata, India. All methods adhered to the applicable guidelines and regulations.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Fish Management and Experimental Design\u003c/h2\u003e\u003cp\u003e Adult zebrafish were sourced from a local ornamental fish breeding center in Naihati, West Bengal, India. The experiments were conducted at the Aquatic Bioresource Research Laboratory, Department of Zoology, University of Calcutta, Kolkata, with prior approval from the University\u0026rsquo;s ethical committee. The fish were bred in the laboratory for two generations before the study. The fish were housed in fiber-reinforced plastic aquaria (2.5 ft x 1 ft x 1 ft) with glass panels on the lateral sides for visibility. The system operated on a flow-through circulatory mechanism, with water filtered through an iron filter near the inlet point. The fish were acclimatized for seven days in dechlorinated tap water, maintained at 25\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, pH 7.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1, salinity 0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 mg/L, dissolved oxygen 6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.5 mg/L, ammonia 1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5 mg/L, and hardness 215\u0026thinsp;\u0026plusmn;\u0026thinsp;25 mg/L. A 14:10 light-dark cycle was maintained. The fish were fed live \u003cem\u003eArtemia nauplii\u003c/em\u003e once daily and vitamin-enriched flake food twice daily ad libitum. The aquarium water was continuously aerated using stone diffusers connected to an electronic air compressor. One-third of the water was replaced with filtered tap water daily, and the aquaria screens were cleaned regularly. A constant concentration of cadmium chloride was maintained to ensure exposure to the heavy metal. Excess food and fecal matter were removed to maintain water quality. The mortality rate during acclimatization was below 1%.\u003c/p\u003e\u003cp\u003eThe sub-lethal exposure experiment lasted 21 days, during which fish were exposed to 0.4 mg/L cadmium (1/10th of the 96-hour LC50 at 26\u0026deg;C) at two temperatures: 26\u0026deg;C (optimal) and 34\u0026deg;C (heat shock). Each experimental group consisted of ten fish, with triplicate setups for each condition. A control group was maintained in unchlorinated, aerated tap water. The test solution was replaced every 48 hours, and fish were fed twice daily. At the end of the exposure period, fish were anaesthetized and then euthanized using MS-222 (tricaine methanesulfonate). Whole-body samples were collected for cadmium accumulation analysis, while liver, ovary, gills, muscle, brain, and alimentary canal tissues were dissected and stored at -80\u0026deg;C for further analysis. Fish samples were rinsed with distilled water, and organs were dissected using stainless steel instruments. For whole-body analysis, fish were kept intact after fin removal.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Rate of bioaccumulation of cadmium in zebrafish\u003c/h2\u003e\u003cp\u003eCadmium bioaccumulation in whole fish and various organs (muscle, gill, ovary, brain, and alimentary canal) was evaluated using Atomic Absorption Spectroscopy (AAS), following the APHA 23rd edition (2017). Fish and dissected organs were digested as described by Bawuro et al (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Prior to digestion, all glassware was soaked in 10% nitric acid for 24 hours, rinsed with distilled water, 0.5% (w/v) potassium permanganate solution, and finally distilled water. A 1 mg sample from each organ was digested with a 1:1 mixture of nitric acid and perchloric acid, followed by sulfuric acid. The mixture was heated at 200\u0026deg;C for 30 minutes, cooled, and then made up to 50 ml with distilled water. Cadmium concentration was determined using an Agilent AA55 Atomic Absorption Spectrophotometer, selecting the appropriate wavelengths for analysis. An analytical blank was prepared in the same manner. Results were reported as \u0026micro;g/g wet weight.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Determination of Metallothionein-2 (MT-2) Concentration, Distribution, and Histopathological Analysis in Fish Tissues\u003c/h2\u003e\u003cp\u003eMetallothionein-2 (MT-2) concentration and distribution were determined using two methods: Enzyme-Linked Immunosorbent Assay (ELISA) and immunofluorescence. For MT-2 quantification, the ELISA method (Wu et al. \u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) was employed using a sandwich kit from Bioassay Technology Laboratory designed for fish MT detection. Whole fish were euthanized, rinsed in PBS, and homogenized on ice. The homogenate was centrifuged, and the supernatant was used for MT-2 quantification. The MT-2 binds to a pre-coated plate with fish MT antibody, followed by biotinylated MT antibody and Streptavidin-HRP. After incubation, unbound Streptavidin-HRP was washed away, and a substrate solution was added to develop color, proportional to MT concentration. The reaction was terminated by adding an acidic stop solution, and absorbance was measured at 450 nm. Results were expressed as ng/ml.\u003c/p\u003e\u003cp\u003eFor tissue distribution, immunofluorescence was used on cryosectioned tissues. Target organs (gills, muscle, and ovary) were dissected, fixed in paraformaldehyde, cryopreserved in sucrose solutions, and embedded in OCT medium. Sections were then immunolabeled with primary and secondary antibodies, followed by visualization under a fluorescent microscope. The distribution of MT-2 was observed in liver, gill, and ovary tissues. Additionally, histopathological analysis was performed on dissected specimens. Organs were processed by cryosectioning, and tissue sections were stained using the Hematoxylin-Eosin technique for permanent slide preparation. Slides were mounted with DPX, and photomicrographs were taken under an Olympus BX51 microscope to identify any abnormalities in tissue architecture.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Assessment of histopathological alterations in Cadmium affected zebrafish liver\u003c/h2\u003e\u003cp\u003eLiver tissues were aseptically dissected on ice and transferred to a Petri dish containing 1X PBS. The samples were processed through Cryosectioning (Campbell et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), with serial sections cut at a thickness of 4\u0026ndash;6 \u0026micro;m. The sections were stained using the Hematoxylin-Eosin technique for permanent slide preparation (Deivasigamani \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Slides were mounted with DPX, and photomicrographs were captured using an Olympus BX 51 compound microscope under appropriate magnification to identify tissue abnormalities.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Evaluation of biochemical and oxidative stress markers in zebrafish liver under Cadmium exposure\u003c/h2\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.6.1 Analysis of biochemical parameters and stress enzyme levels\u003c/h2\u003e\u003cp\u003eThe analysis of biochemical parameters and stress enzyme levels was conducted using specific kits from Precision Biomed Pvt. Ltd, following the methodology of Roy et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). All assays were performed with the Robonik Priest Touch Biochemistry Analyser at various filters. Whole fish were euthanized on ice, with fins and heads ablated. The body was cut into small pieces, rinsed in ice-cold phosphate buffer (PBS, pH 7.4) to remove excess blood, and liver tissue was collected for stress enzyme assays. The tissue was homogenized and sonicated at 4\u0026deg;C in PBS to prepare a homogenate, which was centrifuged at 2000\u0026ndash;3000 RPM for 20 minutes at 4\u0026deg;C. The supernatant was stored at -80\u0026deg;C until further analysis and thawed at 2\u0026ndash;4\u0026deg;C before use.\u003c/p\u003e\u003cp\u003eGlucose concentration (mg/dl) was estimated using the Glucose Oxidase-Peroxidase (GOD-POD) method (Trinder \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e1969\u003c/span\u003e), with absorbance measured at 500\u0026ndash;540 nm. Total protein (g/dl) and direct bilirubin (mg/dl) were assessed via the Biuret method and modified Jendrassik and Grof method (Young \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), both at 546 nm. Cholesterol (mg/dl) was measured with the Cholesterol Oxidase/Peroxidase Aminophenazone i.e., CHOD/PAP method at 510 nm, while calcium (mg/dl) and albumin levels were determined using the Arsenazo III method and BCG method (Young, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1997\u003c/span\u003e), respectively at 630 nm. Triglycerides were analyzed via the Glycerol Phosphate Oxidase (GPO) method (Young \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) at 510 nm. Alkaline Phosphatase (ALP) activity (U/l) was assessed by converting p-Nitrophenyl phosphate to p-Nitrophenol and phosphate, with absorbance measured at 405 nm every 30 seconds for 90 seconds at 37\u0026deg;C. Aspartate Aminotransferase (AST) activity (mU/ml), or Glutamate Oxaloacetate Transaminase, was measured using the International Federation of Clinical Chemistry method without pyridoxal phosphate with absorbance recorded at 340 nm per minute for 180 seconds (Young, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Similarly, Alanine Aminotransferase (ALT) activity (mU/ml), or Glutamate Pyruvate Transaminase, was measured under identical conditions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section3\"\u003e\u003ch2\u003e2.6.2 Assessment of ROS production in zebrafish liver after Cadmium exposure\u003c/h2\u003e\u003cp\u003eThe intracellular ROS production in the liver of zebrafish, in response to cadmium exposure, was evaluated by assessing phagocytic respiratory burst activity using flow cytometric analysis, following the method described by Pal (2020). Single-cell suspensions were prepared from the liver by treating tissue with Collagenase I (2 mg/ml) at 37\u0026ordm;C with continuous shaking. The cells were then stained for 30 minutes with 2'\u0026ndash;7'-Dichlorodihydrofluorescein diacetate (H2-DCFDA), a cell-permeable fluorescent and chemiluminescent probe, in a Ca2+-enriched binding buffer at room temperature in the dark. The ROS generation was measured by analyzing the stained cells using a BD Accuri C6 Flow Cytometer. ROS was detected in the FL1 channel with an emission filter set at 489 nm. The mean fluorescence values of oxidized DCF were recorded and compared between treated and control samples. Data analysis was conducted using BD Accuri C6 software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.6.3 Estimation of Oxidative stress enzyme levels in Liver\u003c/h2\u003e\u003cp\u003eThe in-vitro quantitative estimation of oxidative stress enzyme activity, including Superoxide Dismutase (SOD), Catalase (CAT), Glutathione Peroxidase (GPx), Glutathione Reductase (GR), Glutathione (GSH), and Lipid Peroxidase (LPO) concentrations in the liver of both control and treated fish was conducted using specific ELISA kits (Bioassay Technology Laboratory, Korain Biotech Co., Ltd.), following the protocol described by Roy et al. (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). For sample preparation, liver tissue was aseptically dissected from live, healthy control fish and cadmium- and temperature-treated \u003cem\u003eDanio rerio\u003c/em\u003e. The tissue was homogenized with 1 ml of phosphate buffer solution (PBS, pH 7.4) using an automated homogenizer (Remi Motor, India). The homogenate was centrifuged at 2000\u0026ndash;3000 RPM for 20 minutes, and the supernatant was collected and stored at -20\u0026deg;C for further analysis.\u003c/p\u003e\u003cp\u003eThe assay involved adding 50 \u0026micro;l of standard solution and 50 \u0026micro;l of streptavidin-HRP to the standard solution well. For the sample well, 40 \u0026micro;l of the sample was mixed with 10 \u0026micro;l of the respective antibodies (SOD, CAT, GPx, GR, GSH, or LPO) and 50 \u0026micro;l of streptavidin-HRP. The strips were covered with a seal plate membrane, gently shaken to ensure proper mixing, and incubated at 37\u0026deg;C for 60 minutes in the dark. The plate was washed five times with washing buffer, and 50 \u0026micro;l each of Chromogen Solution A and B were added, followed by a 10-minute incubation at 37\u0026deg;C for color development. The reaction was stopped with 50 \u0026micro;l of stop solution, causing a color change from blue to yellow. The absorbance (OD) of each well was measured at 450 nm. The linear regression equation of the standard curve was calculated based on the standards' concentration and corresponding OD values, and the enzyme activity of each sample was determined accordingly.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Assessment of cell death patterns and mitochondrial membrane potential in zebrafish liver after cadmium exposure\u003c/h2\u003e\u003cp\u003eThe identification of cell death patterns in the liver of \u003cem\u003eDanio rerio\u003c/em\u003e was performed using flow cytometry with Annexin V-FITC and Propidium Iodide (PI) dyes, following the methodology described by Pal (2020). The required chemicals included Fluorescein Isothiocyanate (FITC)-conjugated Annexin V, Propidium Iodide, and Annexin binding buffer. Single-cell suspensions were prepared from liver and gill tissues, stained for 30 minutes at room temperature in the dark with FITC-conjugated Annexin V and PI in a calcium-enriched binding buffer, and analyzed using a BD Accuri C6 Flow Cytometer. For each set, a total of 10,000 events were recorded. Annexin V and PI emissions were detected in the FL1 and FL2 channels with emission filters at 508 nm and 643 nm, respectively. The data were analyzed using BD Accuri C6 Software.\u003c/p\u003e\u003cp\u003eThe mitochondrial membrane potential assay was performed via flow cytometry to evaluate stress-induced apoptotic cell death through mitochondrial depolarization, following Zeng et al (\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e2008\u003c/span\u003e) with minor modifications. JC-1 dye and 1X PBS were used. The cell suspension was centrifuged at 200 g for 5 minutes, and the pellet was resuspended in 1 ml of freshly prepared JC-1 solution. JC-1 accumulates in mitochondria, emitting red fluorescence in cells with intact membrane potential, while apoptotic cells with collapsed membrane potential exhibit green fluorescence (Kumar et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Both control and treated samples were incubated at 37\u0026deg;C for 15 minutes in the dark and analyzed on a BD Accuri C6 flow cytometer with excitation at 488 nm. Green fluorescence was detected at 530 nm (FL1 channel) and red at 570 nm (FL2 channel), with 10,000 events recorded per set. Data analysis was conducted using BD Accuri C6 Software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error (SE). Statistical analyses were performed using one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test to assess differences between experimental groups. A significance level of \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. All statistical analyses were conducted using GraphPad Prism (Version 7.0, GraphPad Software, USA).\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Cadmium Bioaccumulation in Danio rerio\u003c/h2\u003e\u003cp\u003eThe bioaccumulation of cadmium (Cd) was evaluated in whole-body samples (Fig.\u0026nbsp;1a) and specific tissues, including muscle, gills, liver, alimentary canal, and ovary (Fig.\u0026nbsp;1b), following 21 days of exposure to 0.4 mg/L Cd at two different temperatures (i.e., 26\u0026deg;C and 34\u0026deg;C). The whole-body Cd accumulation was significantly influenced by temperature, with the highest Cd concentration recorded in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (96.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.59 \u0026micro;g/g wet weight). This accumulation was significantly higher (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) compared to both the control group (1.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 \u0026micro;g/g wet weight, below the measurable threshold) and the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (74.24\u0026thinsp;\u0026plusmn;\u0026thinsp;2.27 \u0026micro;g/g wet weight). Statistical analysis using Tukey\u0026rsquo;s multiple comparison test further confirmed a significantly higher Cd accumulation (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) in the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group compared to both untreated groups. However, no significant difference was observed between the control and 34\u0026deg;C untreated groups (1.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;g/g wet weight, below the measurable threshold).\u003c/p\u003e\u003cp\u003eOrgan-specific Cd accumulation, analyzed using Atomic Absorption Spectroscopy (Fig.\u0026nbsp;1b), revealed that the liver exhibited the highest Cd bioaccumulation, followed by the ovary, gills, muscle, brain, and alimentary canal (Liver\u0026thinsp;\u0026gt;\u0026thinsp;Ovary\u0026thinsp;\u0026gt;\u0026thinsp;Gills\u0026thinsp;\u0026gt;\u0026thinsp;Muscle\u0026thinsp;\u0026gt;\u0026thinsp;Brain\u0026thinsp;\u0026gt;\u0026thinsp;Alimentary Canal). Among the Cd-exposed groups, the highest Cd concentration was detected in the liver of the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (32.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38 \u0026micro;g/g wet weight), which was significantly higher (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) than in the ovary (29.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.26 \u0026micro;g/g wet weight), gills (17.04\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 \u0026micro;g/g wet weight), muscle (11.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 \u0026micro;g/g wet weight), brain (10.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 \u0026micro;g/g wet weight), and alimentary canal (6.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 \u0026micro;g/g wet weight). In contrast, Cd levels in the liver of the control group (0.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09 \u0026micro;g/g wet weight) and the 34\u0026deg;C untreated group (0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 \u0026micro;g/g wet weight) remained below threshold limits. However, a significant increase (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.00001) in liver Cd content was observed in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group compared to the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (28.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18 \u0026micro;g/g wet weight), with a similar trend observed in all other tissues. The Cd concentrations in different organs are summarized in Supp. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Tukey\u0026rsquo;s multiple comparison test further revealed a significant difference (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in Cd bioaccumulation between the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd and 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd groups across all analyzed tissues.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSemi-quantitative scoring of histopathological alterations in the liver of \u003cem\u003eDanio rerio\u003c/em\u003e after 21 days of exposure to 0.4 mg/l Cd at two temperatures (26\u0026deg;C and 34\u0026deg;C). The severity of lesions is graded as follows: (-) none, (+) mild, (++) moderate, and (+++) severe.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCondition\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e26\u0026deg; Control\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e34\u0026deg; Control\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e26\u0026deg; Treated\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e34\u0026deg; Treated\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1. Blood congestion in sinusoid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e+\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2. Swelling of hepatocyte\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3. Presence of dark granules\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e4. Cellular degeneration\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e+++\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e++\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Metallothionein concentration as a biomarker for cadmium-induced stress in Danio rerio\u003c/h2\u003e\u003cp\u003eThe concentration of cellular stress protein, Metallothionein is a good indicator of water pollution and also reported as a good bioindicator of heavy metals pollution. Metallothioneins (MT) are cysteine rich proteins able to sequester Cd ions. Since MT is found in several organs like liver, kidney, muscle etc., we have determined the concentration in whole organism (Fig.\u0026nbsp;2a). The results indicated that, MT concentration increases synchronously with Cd accumulation rates and likewise the highest level was found in 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group of fish. Dunnett\u0026rsquo;s Multiple comparison test revealed that, there is a significant difference (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) between control group (16.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.09 ng/ml) and 34\u0026deg;C treated group (25.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.19 ng/ml) which indicates that temperature has a potential effect on MT concentration. The Cd treated groups both at 26\u0026deg;C (67.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.96 ng/ml) and 34\u0026deg;C (67.06\u0026thinsp;\u0026plusmn;\u0026thinsp;1.9 ng/ml) showed significant difference (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e3.2.1 Immunofluorescence localization of metallothionein in liver of Danio rerio\u003c/h2\u003e\u003cp\u003eImmunopositivity reactions for Metallothionein (MT) was determined in the cells of liver (Fig.\u0026nbsp;2b) for all four experimental groups. Immunofluorescence analysis revealed substantial localization of MT in the tissues of Cd treated groups. The immunopositivity for MT protein showed an increase in groups treated with Cd at both 26\u0026deg;C and 34\u0026deg;C but the untreated groups showed basal level of MT expression. While at the most intense MT protein expression was observed in group treated with both Cd and high temperature (34\u0026deg;C). The immunodetection for MTs shows the absence of signal in the tissues of the animals from the control group. The liver tissues showed a marked increase in the Immunoreactivity of MTs in the Cd treated group at 34\u0026deg;C compared to the one at optimum temperature (26\u0026deg;C). MT was found to be distributed both in the cytoplasmic and nuclear compartments of the hepatocytes (Fig.\u0026nbsp;2b). Since, the hepatocytes are tightly packed, strong immunopositivity is found among the Cd treated groups (Fig.\u0026nbsp;2b). No immunofluorescence was found in the untreated groups of fishes.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Histopathological analysis of cadmium-induced alterations in Danio rerio liver tissue\u003c/h2\u003e\u003cp\u003eA light microscopic analysis of cadmium affected \u003cem\u003eDanio rerio\u003c/em\u003e liver tissue revealed distinct structural features across experimental groups (Fig.\u0026nbsp;3). The control group (26\u0026deg;C) exhibited a normal liver architecture with polyhedral hepatocytes (HS), centrally located nuclei, and prominent nucleoli (Fig.\u0026nbsp;3A). Blood sinusoids (BS), lined with reticulo-endothelial cells, were surrounded by hepatocytes, while bile canaliculi were found near the portal vein, lined with simple cuboidal epithelium. Lipid glycogen granules were also present in the cytoplasm (Fig.\u0026nbsp;3A). Exposure to elevated temperature (34\u0026deg;C) resulted in mild blood congestion in the sinusoids and hydropic swelling of hepatocytes, though the nuclei retained a nearly normal shape. A substantial number of normal hepatocytes were observed in this group (Fig.\u0026nbsp;3B).\u003c/p\u003e\u003cp\u003eCd exposure at both optimum (26\u0026deg;C) and high (34\u0026deg;C) temperatures led to the accumulation of dark granules in some hepatocytes (Fig.\u0026nbsp;3C and D). Histopathological alterations included degenerated hepatic tissue, vacuole formation, blood cell infiltration, cellular swelling, nuclear degeneration, karyorrhexis, loss of hepatocytic cell walls, and disorganized hepatic cords. The liver morphology became fragmented, with no compact structure as seen in the control group. The 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group exhibited numerous lipid droplets, dark granule accumulation, loss of hepatocyte polyhedral architecture, and lobular disruption. Fat degeneration resulted in irregular clear spaces between hepatocytes, along with mild hypertrophy and widened sinusoids (Fig.\u0026nbsp;3D). A Semi-Quantitative Scoring was performed to assess the relative severity of histopathological lesions, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Biochemical alterations in liver tissue after cadmium exposure and elevated temperature in Danio rerio\u003c/h2\u003e\u003cp\u003eChanges in the biochemical parameters of Control, 34\u0026deg;C treated and Cd treated at 26\u0026deg;C and 34\u0026deg;C are summarized in Fig.\u0026nbsp;4. After 21 days of chronic Cd exposure significant changes were found in the liver tissue for all parameters i.e. Glucose, Total protein, Cholesterol, Bilirubin, Triglyceride and Calcium. Tukey\u0026rsquo;s multiple comparison test revealed that, glucose level had increased significantly (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001, df\u0026thinsp;=\u0026thinsp;3) in groups exposed to Cd as well as a combination of higher temperature with cadmium compared to control fishes. Whereas, no significant difference was found among the 26\u0026deg;C and 34\u0026deg;C untreated groups (Fig.\u0026nbsp;4a). The highest value of glucose was found in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd treated group (224.4\u0026thinsp;\u0026plusmn;\u0026thinsp;8.75 mg/dl) followed by 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd treated group (63.24\u0026thinsp;\u0026plusmn;\u0026thinsp;1.99 mg/dl), 34\u0026deg;C group (41.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21 mg/dl) and Control fishes (27.61\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54 mg/dl).\u003c/p\u003e\u003cp\u003eA significant reduction in Total Protein, Cholesterol, and Triglyceride levels was observed in cadmium-exposed groups compared to the control (Fig.\u0026nbsp;4b, 4c, 4d). Tukey\u0026rsquo;s multiple comparison test showed a notable decline in Total Protein levels (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001, df\u0026thinsp;=\u0026thinsp;3), with the lowest value in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (0.644\u0026thinsp;\u0026plusmn;\u0026thinsp;0.018 g/dl), followed by the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (0.735\u0026thinsp;\u0026plusmn;\u0026thinsp;0.021 g/dl). Cholesterol levels significantly dropped in Cd-treated groups with the lowest in the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (9.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 mg/dl), followed by 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd (13.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 mg/dl). Triglyceride levels also decreased significantly, with the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group recording the lowest (47.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.66 mg/dl), which was not significantly different from the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (49.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73 mg/dl). Bilirubin levels significantly increased in all Cd-treated groups, with the highest concentration in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (7.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.22 mg/dl) (Fig.\u0026nbsp;4e). Calcium levels decreased significantly across all treated groups, with the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group showing the lowest (1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.069 mg/dl) (Fig.\u0026nbsp;4f).\u003c/p\u003e\u003cp\u003eCorrelation analysis based on Pearson\u0026rsquo;s correlation coefficient (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was performed among biochemical parameters with an aim to find linear relation between analyzed parameters. Further, heatmap was used to visualize hierarchical clustering among the biochemical parameters (using GraphPad Prism 8.0.1). According to Pearson\u0026rsquo;s correlation coefficient, a strong positive correlation was found between Glucose and Bilirubin content (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.870) (Supp. Figure\u0026nbsp;1). Total protein was positively correlated with Cholesterol (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.826), Triglyceride (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.930) and Calcium content (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.703). Similarly, Cholesterol content was found to be positively correlated with Triglyceride (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.880) and Calcium (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.917). Although a strongly negative correlation was found between Total protein and Bilirubin (\u003cem\u003er\u003c/em\u003e = -0.877); Bilirubin and Triglyceride (\u003cem\u003er\u003c/em\u003e = -0.819).\u003c/p\u003e\u003cdiv id=\"Sec20\" class=\"Section3\"\u003e\u003ch2\u003e3.4.1 Oxidative stress response through ROS in Liver\u003c/h2\u003e\u003cp\u003eCadmium exposure significantly (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) increased ROS production in all treated groups compared to the control (Supp. Figure\u0026nbsp;2). The highest ROS level was observed in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (62950\u0026thinsp;\u0026plusmn;\u0026thinsp;86.13), significantly higher than the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (48704\u0026thinsp;\u0026plusmn;\u0026thinsp;163.9). Notably, the 34\u0026deg;C Cd-untreated group (38671\u0026thinsp;\u0026plusmn;\u0026thinsp;204.1) also exhibited elevated ROS levels compared to the control (25666\u0026thinsp;\u0026plusmn;\u0026thinsp;107.6), indicating temperature-induced oxidative stress.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section3\"\u003e\u003ch2\u003e3.4.2 Evaluation of antioxidant and stress enzyme activities in liver\u003c/h2\u003e\u003cp\u003eSOD and CAT activities were significantly elevated in Cd-exposed groups (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;5a, 5b), with the highest levels observed in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (SOD: 158.9\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02 U/mg Protein/min; CAT: 43.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37 U/mg Protein/min). No significant difference was found between the 34\u0026deg;C Cd-untreated and control groups for CAT activity (Fig.\u0026nbsp;5b). Furthermore, \u003cem\u003eGSH\u003c/em\u003e, \u003cem\u003eGPx\u003c/em\u003e, and \u003cem\u003eGR\u003c/em\u003e activities were also significantly increased in Cd-treated groups (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;5c-e). The 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group exhibited the highest enzyme activities (\u003cem\u003eGSH\u003c/em\u003e: 12.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.29 nmole/mg Protein/min; GPx: 21.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.36 nmole/mg Protein/min; GR: 16.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28 nmole/mg Protein/min), significantly higher than the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group and controls. Tukey\u0026rsquo;s test indicated no significant difference in GPx activity between the 34\u0026deg;C Cd-untreated and control groups, though GSH and GR levels were significantly different.\u003c/p\u003e\u003cp\u003eCadmium exposure significantly increased ALP, AST, and ALT concentrations (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Fig.\u0026nbsp;5f-h). The 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group showed the highest ALP levels (8.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.16 U/L), significantly greater than the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (6.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 U/L) and controls. AST and ALT levels followed a similar trend, with significant increases in treated groups at both temperatures compared to controls. LPO levels increased substantially in Cd-treated groups (Fig.\u0026nbsp;5i), with the highest concentration in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (56.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.46 nmole/ml), significantly exceeding the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (41.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32 nmole/ml) and controls. Notably, LPO levels in the 34\u0026deg;C untreated group were also significantly (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001) higher than controls\u003c/p\u003e\u003cp\u003eAccording to the correlation analysis based on Pearson\u0026rsquo;s correlation coefficient a very strong positive correlation was found among the antioxidant enzymes with the stress enzymes. SOD has strong positive correlation with all the stress parameters except GPx (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.736), similarly CAT shown very low positive correlation with GPx (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.360) as well. A very strong positive correlation was found between GSH, GPx and GR whereas LPO was found to be more positively correlated with ALP, AST and ALT (\u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.994, \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.971 and \u003cem\u003er\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.992 respectively).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Apoptotic Analysis in Liver Tissue of Danio rerio: Annexin V-FITC/PI Staining and MMP Assay\u003c/h2\u003e\u003cp\u003eAnnexin V-FITC/PI staining analysis demonstrated apoptosis induction in the liver of \u003cem\u003eDanio rerio\u003c/em\u003e across four experimental groups (Fig.\u0026nbsp;6a; Supp. Figure\u0026nbsp;3a-d). Double-negative staining indicates live cells (LL), Annexin V-FITC positive and PI negative staining represents early apoptosis (LR), and double-positive staining indicates late apoptosis (UR) (Supp. Figure\u0026nbsp;3a-d). Tukey\u0026rsquo;s Multiple Comparison Test revealed a significant increase (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in both early and late apoptotic cells in Cd-treated groups compared to the control. The percentage of normal cells significantly declined in treated groups (Fig.\u0026nbsp;6a). The highest percentage of early apoptotic cells was observed in the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (18.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38), followed by the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (15.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31), 34\u0026deg;C untreated group (10.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37), and control group (6.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37), with a significant difference (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) between treated and control groups. For late apoptotic cells, a significant increase was observed in the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd (1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10) and 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd (4.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21) groups compared to the control (0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), while no significant difference was found between the 34\u0026deg;C untreated group (0.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) and the control.\u003c/p\u003e\u003cp\u003eThe MMP assay in the liver of \u003cem\u003eDanio rerio\u003c/em\u003e (Fig.\u0026nbsp;6b; Supp. Figure\u0026nbsp;3e-h) revealed that the disruption of active mitochondria, a hallmark of early apoptosis, leads to changes in membrane potential. In healthy cells, JC-1 accumulates in mitochondria, forming J-aggregates with red fluorescence. However, in apoptotic cells with reduced MMP, JC-1 remains in monomeric form, emitting green fluorescence. In the control group, most cells maintained normal MMP (ΔΨm), showing bright fluorescence in both BL1-A and BL3-A channels (BL1-A bright, BL3-A bright) (Supp. Figure\u0026nbsp;3e and f). In contrast, cells in both temperature- and Cd-treated groups exhibited reduced BL3-A intensity (BL1-A bright, BL3-A dim), indicating compromised ΔΨm (Supp. Figure\u0026nbsp;3g and h). The collapse of ΔΨm is associated with mitochondrial depolarization, which can trigger cytochrome c release and apoptosis. The percentage of polarized cells significantly decreased (\u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the treatment groups, with the lowest percentage observed in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (Fig.\u0026nbsp;6b). Additionally, an increase in depolarized cells in the 34\u0026deg;C untreated group suggests that elevated temperature alone can induce cell death. The percentage of cells with polarized MMP was significantly lower in the Cd-treated groups (26\u0026deg;C, 74.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39; 34\u0026deg;C, 68.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77) compared to the control (86.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27) and 34\u0026deg;C untreated group (80.47\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42). The highest percentage of depolarized cells was found in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (31.37\u0026thinsp;\u0026plusmn;\u0026thinsp;0.76), followed by the 26\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (25.74\u0026thinsp;\u0026plusmn;\u0026thinsp;0.39), 34\u0026deg;C untreated group (19.53\u0026thinsp;\u0026plusmn;\u0026thinsp;0.42), and control (13.75\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27).\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eCd bioaccumulation in aquatic environments poses a significant risk to human health through fish consumption. Key factors influencing Cd accumulation in fish tissues include environmental concentration, exposure duration, temperature, and pH. This study demonstrated a tissue-specific accumulation pattern, with the liver exhibiting the highest concentration (Fig.\u0026nbsp;1b) due to its role in detoxification and metabolic functions. The order of accumulation was Liver\u0026thinsp;\u0026gt;\u0026thinsp;Ovary\u0026thinsp;\u0026gt;\u0026thinsp;Gills\u0026thinsp;\u0026gt;\u0026thinsp;Muscles\u0026thinsp;\u0026gt;\u0026thinsp;Brain\u0026thinsp;\u0026gt;\u0026thinsp;Alimentary canal. Elevated temperatures further amplified Cd accumulation, especially in the liver and ovaries. The gills, serving as the primary entry point for Cd due to their extensive surface area and vascular structure, also showed substantial accumulation (Fig.\u0026nbsp;1b). In contrast, the brain and muscles had lower Cd levels due to the protective blood-brain barrier and limited detoxification activity. The study highlights that both Cd exposure and elevated temperatures significantly influence bioaccumulation and toxicity in \u003cem\u003eDanio rerio\u003c/em\u003e. Waterborne Cd can cause oxidative stress, immunosuppression, and endocrine disruption, ultimately affecting growth and survival (Cao et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kumar and Singh \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Wen et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The observed temperature-dependent increase in Cd accumulation, particularly in the liver, underscores the synergistic effect of elevated temperature on metal retention. These findings align with previous research showing that higher temperatures enhance the uptake and retention of toxic metals in aquatic organisms (Wen et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Moiseenko et al. 2020; Castaldo et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eMT is known to sequester heavy metals, thereby protecting cells from toxicity, but its elevated levels also reflect the burden of Cd exposure in the organism (Ruttkay-Nedecky et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This study confirms that exposure to metals usually promotes the induction of MT (Fig.\u0026nbsp;2a), which is consistent with the previous studies (Shariati et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wen et al. \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition to increased Cd bioaccumulation, co-exposure to Cd resulted in an elevated MT concentration in \u003cem\u003eD. rerio\u003c/em\u003e (Lu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Such distinct results indicate that MT induction is positively correlated with the accumulation of Cd in fish tissue. The significant correlation between Cd levels and MT concentrations further supports the role of MT as a reliable biomarker for Cd exposure and toxicity.\u003c/p\u003e\u003cp\u003eHistological analysis serves as a valuable biomarker for assessing the impact of xenobiotic compounds on biological systems (Paolini et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). In this study, cadmium-induced histopathological alterations in the liver, including cellular swelling, vacuole formation, and disrupted hepatic architecture, align with previous findings on cadmium's hepatotoxicity in fish (Chavan and Muley \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Kaur et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Severe lesions such as lymphonuclear cell infiltration, degeneration of hepatic parenchyma, and hepatocyte deformation were observed in \u003cem\u003eL. rohita\u003c/em\u003e exposed to heavy metals (Kaur et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Similar pathological changes, including cytoplasmic vacuolation and focal necrosis, were reported in \u003cem\u003eC. mrigala\u003c/em\u003e exposed to lead acetate (Chavan and Muley \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Additionally, \u003cem\u003eTilapia mossambica\u003c/em\u003e exhibited severe damage and vacuolation after exposure to cadmium sulfate (Jalaludeen et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Cadmium exposure also caused hypertrophy, vacuolation, and necrosis in \u003cem\u003eCyprinus carpio\u003c/em\u003e (Patnaik et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), while \u003cem\u003eC. mrigala\u003c/em\u003e showed complete disintegration of liver tissue when exposed to lead nitrate (Mary et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). These findings highlight the liver\u0026rsquo;s role as a primary detoxification site and the accumulation of cadmium bound to metallothionein, leading to prolonged histopathological damage (Olsson and Hogstrand \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1987\u003c/span\u003e). Such structural changes serve as crucial indicators of heavy metal toxicity in aquatic environments.\u003c/p\u003e\u003cp\u003eThe rise in glucose levels in \u003cem\u003eDanio rerio\u003c/em\u003e exposed to Cd and high temperatures (Fig.\u0026nbsp;4a) indicates metabolic disruption, likely due to impaired liver function. This increase stems from gluconeogenesis, which supplies energy for detoxification (Zutshi et al. \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Kavitha et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and is driven by stress hormones like cortisol and catecholamines that trigger glycogenolysis (Randall and Ferry \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e1992\u003c/span\u003e). Similar patterns were observed in \u003cem\u003eMugil cephalus\u003c/em\u003e (Hilmy et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1987\u003c/span\u003e), while \u003cem\u003eClarias batrachus\u003c/em\u003e exhibited reduced glucose levels when exposed to Cd and Hg (Arya \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The findings suggest that Cd-induced stress, amplified by elevated temperatures, elevates glucose as an adaptive energy response.\u003c/p\u003e\u003cp\u003eThe observed reduction in protein content in Cd-exposed fish (Fig.\u0026nbsp;4b) may result from impaired protein synthesis or increased protein breakdown due to metabolic stress, possibly from nephrosis or cirrhosis (Arya \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Excessive proteolysis is likely occurring to meet energy demands during Cd exposure, as reflected by elevated glucose levels. Similar protein reductions have been reported in various fish species exposed to heavy metals (Cicik and Engin \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Binukumari et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Additionally, increased protease activity and proteolysis contribute to energy production during Cd stress. Unconjugated bilirubin, a product of heme catabolism, showed a significant increase in Cd-exposed groups, particularly at higher temperatures. This elevation could indicate hemolysis or impaired bilirubin conjugation. Elevated bilirubin levels are commonly associated with stress responses (Levitt and Levitt \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), although conjugation in zebrafish is not well studied (Liu et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2013\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eA decrease in cholesterol levels was observed in the Cd-exposed fish (Fig.\u0026nbsp;4c), possibly due to kidney tissue damage and membrane disruption. Conversely, an increase in cholesterol was reported in \u003cem\u003eO. niloticus\u003c/em\u003e (\u0026Ouml;ner et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The reduction in cholesterol suggests environmental stress and increased lipid utilization to meet energy demands. Similarly, a decline in triglycerides was noted, likely due to impaired lipid metabolism and glycogen storage (Heydarnejad et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Calcium levels were significantly lower in the Cd-exposed groups (4f), with the greatest reduction at elevated temperatures. Similar declines were observed in other species exposed to Cd (Honda and Suzuki \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Reduced calcium may disrupt vitellogenin (VTG) transport and ovarian maturation in \u003cem\u003eD. rerio\u003c/em\u003e, as calcium has a positive correlation with VTG, particularly during yolk deposition (Linares-Casenave et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe correlation matrix reveals a significant negative correlation between glucose and total protein (Supp. Figure\u0026nbsp;1), consistent with Gagnon et al. (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2006\u003c/span\u003e), who observed similar trends in fish under metal stress. This suggests that elevated glucose levels may be linked to liver damage and renal dysfunction (Pratap and Wendelaar Bonga \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e1990\u003c/span\u003e), as supported by the positive correlation between glucose and bilirubin. A study on \u003cem\u003eCyprinus carpio\u003c/em\u003e by Cicik and Engin (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e) showed similar interactions. Additionally, total protein levels were positively correlated with triglycerides and cholesterol, indicating liver dysfunction, reduced absorption, and protein loss (Heydarnejad et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), but showed a negative correlation with bilirubin. The positive correlation between calcium and cholesterol aligns with Linares-Casenave et al (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), highlighting the role of cholesterol synthesis in VTG production.\u003c/p\u003e\u003cp\u003eOxidative stress, as evidenced by increased ROS levels and enhanced antioxidant enzyme activities, is a critical mechanism underlying cadmium toxicity. The 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group exhibited the highest ROS production, indicating that elevated temperature exacerbates oxidative damage. Similar findings have been reported in other studies, where temperature increases led to enhanced oxidative stress in fish exposed to pollutants (Vergauwen et al. \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The SOD-CAT system is usually regarded as the first line of defense towards the production of ROS under environmental stressors (Pandey et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). The superoxide radical can be turned into H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e by SOD, while H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is metabolized by CAT. In this study, Cd led to CAT activation in zebrafish (Fig.\u0026nbsp;5b), probably due to the antioxidative response to elevated H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e production (Lu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). GSH, a vital non-enzymatic antioxidant, plays a crucial role in fish immune functions. It reacts with ROS to form glutathione disulfide and serves as a substrate in detoxifying exotic toxic chemicals through biotransformation catalyzed by glutathione S-transferase (Zhang et al. \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Glutathione reductase (GR) is responsible for maintaining cellular redox balance by regulating the GSH/GSSG ratio (Sarkar et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The observed changes in antioxidant enzyme activities, such as increased SOD, CAT, and GSH activities, reflect the organism's response to mitigate oxidative damage (Company et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Banni et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). However, the reduced efficiency of the antioxidant defence system at higher temperatures suggests an overwhelmed protective response under these conditions.\u003c/p\u003e\u003cp\u003eCd-induced apoptosis, a critical response to cellular damage, was confirmed through Annexin V-FITC/PI staining and mitochondrial membrane potential (MMP) assays. The study revealed a significant increase in early and late apoptotic cells, especially in the 34\u0026deg;C\u0026thinsp;+\u0026thinsp;Cd group (Fig.\u0026nbsp;6a), indicating that elevated temperature enhances cadmium-induced cell death via mitochondrial dysfunction. This aligns with previous findings on cadmium\u0026rsquo;s ability to disrupt MMP and trigger apoptosis (Zhang et al. \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Cadmium exposure leads to oxidative stress, DNA damage, and mitochondrial dysfunction, which activates the intrinsic apoptotic pathway through the release of cytochrome c and caspase activation (Jiang and Wang \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Hosseini et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The elevated production of ROS impairs mitochondrial membrane permeability, resulting in apoptosis. The current study demonstrated that cadmium exposure significantly increased apoptotic hepatocytes and MMP depolarization in \u003cem\u003eD. rerio\u003c/em\u003e, with higher apoptotic rates observed at elevated temperatures. These findings are consistent with prior research on heavy metal-induced apoptosis in fish and other organisms (Gao et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Liu et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The results highlight the critical role of mitochondrial dysfunction in cadmium-induced hepatic apoptosis and the amplifying effect of temperature on this process.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study provides compelling evidence that elevated temperature significantly exacerbates the toxic effects of cadmium in \u003cem\u003eDanio rerio\u003c/em\u003e, leading to increased bioaccumulation across various organs, including muscle, gill, ovary, brain, and alimentary canal. The atomic absorption spectroscopy analysis revealed a substantial rise in cadmium concentration, particularly in metabolically active tissues like the liver. Elevated temperature not only intensified cadmium accumulation but also triggered heightened oxidative stress, as indicated by increased levels of reactive oxygen species and antioxidant enzyme activity. Histopathological examination showed severe tissue damage, including cellular degeneration, necrosis, and vacuolization, while apoptotic markers confirmed enhanced cell death in liver tissue. These findings underscore the synergistic impact of temperature and cadmium exposure, highlighting the vulnerability of aquatic organisms to heavy metal toxicity in the context of climate change. Moreover, this study emphasizes the need for future research to elucidate the molecular pathways involved in temperature-mediated metal toxicity and to develop effective mitigation strategies to safeguard aquatic ecosystems from the dual threats of global warming and heavy metal contamination.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors are thankful to the Head of the Department of Zoology, University of Calcutta, India for providing the facilities. The authors are also thankful to the Director, Zoological Survey of India.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was funded by Council of Scientific \u0026amp; Industrial Research, Senior Research Fellowship (Direct) Programme, Government of India [Sanction No. 09/028(1036)/2018-EMR-I Dated: 16.04.2018]. The authors are also grateful to DST SERB NPDF scheme [Sanction No. PDF/2023/000069] for funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDR and MM: Conceptualization, data curation, methodology, writing - original draft and editing); SP, AM and JKS: review and editing and SHC (conceptualization, supervision, project administration, editing).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests that could have appeared to influence the work reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures involving zebrafish were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Calcutta, Kolkata, India. All methods adhered to the applicable guidelines and regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eI, Roy, D., the corresponding author of this article hereby confirms that all coauthors have agreed on the final version of this manuscript to be published according to the journal guidelines.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Tawwab M, Wafeek M (2017) Fluctuations in water temperature affected waterborne cadmium toxicity: hematology, anaerobic glucose pathway, and oxidative stress status of Nile tilapia, Oreochromis niloticus (L.). 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Annals of Clinical Biochemistry 34(6): 579-581.\u003c/li\u003e\n\u003cli\u003eZeng Y, Cheng H, Jiang X, Han X (2008) Endosomes and lysosomes play distinct roles in sulfatide-induced neuroblastoma apoptosis: potential mechanisms contributing to abnormal sulfatide metabolism in related neuronal diseases. Biochem J 410(1): 81-92.\u003c/li\u003e\n\u003cli\u003eZhang D, Zhang T, Liu J, Chen J, Li Y, Ning G, Huo N, Tian W, Ma H (2019) Zn supplement-antagonized cadmium-induced cytotoxicity in macrophages in vitro: involvement of cadmium bioaccumulation and metallothioneins regulation. J Agric Food Chem 67(16): 4611-4622.\u003c/li\u003e\n\u003cli\u003eZhang Y, Liu J, Zhou Y, Gong T, Wang J, Ge Y (2013) Enhanced phytoremediation of mixed heavy metal (mercury)\u0026ndash;organic pollutants (trichloroethylene) with transgenic alfalfa co-expressing glutathione S-transferase and human P450 2E1. 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Environ Monit Assess 168(1): 11\u0026ndash;19.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Toxicology](https://link.springer.com/journal/44339)","snPcode":"44339","submissionUrl":"https://submission.springernature.com/new-submission/44339/3","title":"Discover Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bioaccumulation, Temperature stress, Metal pollution, Metallothionein, Oxidative stress, Apoptosis","lastPublishedDoi":"10.21203/rs.3.rs-7063175/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7063175/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eElevated environmental temperatures associated with climate change may potentiate heavy metal toxicity in aquatic ecosystems, yet the mechanisms underlying this interaction remain poorly characterized. This study elucidates how temperature modulates cadmium (Cd) bioaccumulation kinetics and subsequent cellular pathophysiology in adult zebrafish (\u003cem\u003eDanio rerio\u003c/em\u003e) during chronic exposure (21 days) at control (26\u0026deg;C) versus elevated (34\u0026deg;C) temperatures. Tissue-specific analysis revealed pronounced hepatic Cd accumulation that was significantly amplified (2.4-fold increase) at 34\u0026deg;C compared to 26\u0026deg;C. This temperature-dependent bioaccumulation pattern corresponded with differential metallothionein induction profiles. Histopathological assessment documented progressive hepatocellular deterioration characterized by cytoplasmic vacuolation, sinusoidal dilation, and leukocyte infiltration\u0026mdash;effects exacerbated at elevated temperature. Comprehensive biochemical profiling demonstrated marked dysregulation of glucose homeostasis, protein metabolism, lipid parameters, and calcium regulation, with temperature-dependent perturbation patterns. Mechanistic investigations revealed that high temperature synergistically enhanced Cd-induced oxidative stress, evidenced by elevated reactive oxygen species generation, lipid peroxidation, and compensatory antioxidant enzyme modulation. Flow cytometric analysis using Annexin V-FITC/PI and JC-1 staining confirmed that temperature amplified Cd-induced hepatocyte apoptosis through mitochondria-dependent pathways. These findings establish temperature as a critical determinant of Cd toxicokinetics and toxicodynamics in fish, with important implications for ecological risk assessment in thermally fluctuating aquatic environments under climate change scenarios.\u003c/p\u003e","manuscriptTitle":"Temperature amplifies cadmium toxicity through bioaccumulation dynamics and hepatic cellular responses in Danio rerio","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-19 14:07:05","doi":"10.21203/rs.3.rs-7063175/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-05T08:23:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-31T20:52:17+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-29T03:24:42+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-27T09:15:13+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"13984699496686863008092847262398922967","date":"2025-08-20T07:27:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"87773387868751759871840780709493612271","date":"2025-08-19T15:31:48+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"123911911692110624120759763014115818407","date":"2025-08-19T13:52:58+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"221313987470172714394923444009658323948","date":"2025-08-12T12:04:04+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-11T14:05:54+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-08T11:27:45+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-06T11:19:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-25T11:29:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Toxicology","date":"2025-07-25T08:58:39+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-toxicology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Toxicology](https://link.springer.com/journal/44339)","snPcode":"44339","submissionUrl":"https://submission.springernature.com/new-submission/44339/3","title":"Discover Toxicology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ebf10d53-b1c5-41b2-9a69-0b2a41936a30","owner":[],"postedDate":"August 19th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-11-04T06:38:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-19 14:07:05","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7063175","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7063175","identity":"rs-7063175","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}