Introduction

Analyzing the toxicity and bioaccumulation of chemicals in the aquatic environment is critical for assessing their environmental risks and hazards. Bioaccumulation studies in aquatic organisms are used to evaluate a compound's behavior in the environment, with bioaccumulation resulting from the absorption, distribution, metabolism, and excretion (ADME) process (Nichols et al. 2007).

Over the last three decades, the Caspian Sea has faced numerous environmental challenges, including oil pollution. Oil contaminants are distributed throughout the Caspian Sea, largely due to petroleum plants located along its shoreline. Massive releases of oil products by Russian and Azerbaijani petroleum companies have exposed the western coasts to significant amounts of these contaminants. Additionally, a large amount of oil deposits, approximately 35 billion barrels, have been discovered in the Caspian Sea area close to Kazakhstan in the last two decades. The enclosed nature of this ecosystem has intensified Caspian Sea pollution (Holstein et al. 2018).

Over a hundred hydrocarbon compounds can be classified as polycyclic aromatic hydrocarbons (PAHs), each containing at least two benzene rings in its chemical structure (Behera et al. 2018; Zhu et al. 2017). Many species are threatened by these perilous materials in both terrestrial and aquatic ecosystems (Yavar Ashayeri et al. 2018). The Caspian Sea PAH concentrations in 2014 were 3228 ng/g dry weight (Yancheshmeh et al. 2014). According to the Environmental Protection Agency (EPA) of the United States, 16 PAHs, including BaP, are carcinogenic (Perrichon et al. 2014; Yan et al. 2004). BaP is the prototypical form of five-ringed PAHs, which are known for their developmental toxicity, reproductive toxicity, immunotoxicity, and carcinogenicity, as well as genotoxic, mutagenic, teratogenic, antioxidant system disruptors, and AChE enzyme inhibitors for aquatic organisms (Esmaeilbeigi et al. 2021; Seemann et al. 2015). Despite its relatively low toxicity, the compound's primary and secondary metabolites generated by biotransformation are highly genotoxic. CYP450 enzymes mediate phase I biotransformation of BaP, yielding highly reactive metabolic intermediates such as 3-hydroxybenzo[a]pyrene (3-OH-BaP) (Rey-Salgueiro et al. 2011; dos Santos et al. 2014). Since the 1980s, atmospheric concentrations of BaP have increased due to anthropogenic activities such as petroleum use, exhaust fumes, spills, and shipping. Various studies have reported different concentrations of BaP in water, particularly in the heavily polluted Caspian Sea (Esmaeilbeigi et al. 2021; Langan et al. 2018). Coastal sediments in the Caspian Sea have shown a variation in BaP concentration ranging from 8.2 to 179.2 ng/g dry weight. One study found that for five types of sturgeon living in the southern Caspian Sea, 0.18 gr/kg of dry tissue penetrated into the tissues for every 130 gr/l of seawater containing PAHs (Khoshbavar-Rostami Soltani et al. 2012).

Persian sturgeon live primarily on beach bottoms in the central and southern Caspian Sea, especially along the seacoast of Iran (Persia) (Moghim et al. 2006; Vavrek et al. 2014). A wide range of factors contribute to the plight of Caspian sturgeon populations. These factors include overfishing, increased water pollution, reduced natural fish reduplication due to blockage of migration routes, and loss of natural reproductive grounds. Approximately 85% of sturgeon have been classified as critically endangered (Moghim et al. 2006; Prokopchuk et al. 2016). Acipenser ruthenus (sterlet) is one of the smallest and most endangered species of sturgeon (Lenhardt et al. 2006; Stanic et al. 2006).

Fish stressors activate a variety of stress reactions at the cellular level. One of the most prominent mechanisms protecting cells from various stressors is the response to thermal shock, which leads to HSP synthesis (Amani et al. 2022; Jahangirizadeh et al. 2020). Prestressing or enhancing HSP receptor expression or preactivating the cellular stress response may reduce stress's lethal effects and reduce stress-induced death in fish (Akbary Moghaddam et al. 2021; Roberts et al. 2010). There are naturally occurring or synthetic compounds that increase HSP expression. By stimulating HSP transcription factors, they induce HSP production (Otaka et al. 2007; Pirali et al. 2020). One such compound, Pro-Tex®, is a resistant precursor that induces HSPs in organisms such as Persian sturgeon (Vahdatiraad et al. 2023a). Pro-Tex® is the soluble form of TEX-OE, a substance extracted from Opuntia ficus indica or Nopal cactus. AMG is another HSPi that is frequently used (Bolarinwa et al. 2014). A number of studies have demonstrated that pyrazole-derived compounds can activate the HSF1 gene (heat shock transcription factor 1) to upregulate HSP70 expression (Brough et al. 2005; Vahdani et al. 2019). Pyrazole is a five-membered, two-nitrogen-containing heterocyclic ring. A variety of derived compounds have been developed based on these structures. Therefore, many researchers have synthesized and evaluated these compounds as therapeutic agents (Küçükgüzel and ŞenkardeŞ 2015).

A continuous cell line can be used to study toxicology, carcinogenesis, and genetic expression and regulation (Lakra et al. 2011). Cell-based in vitro models of fish liver, gills, and kidney that simulate all the body's structural, absorption, detoxification, and other pathophysiological properties have the potential to accelerate toxicity screening by providing a platform with high throughput for assessing ecotoxicity and providing compounds with potential. In accordance with the "3Rs" principles (replacement, reduction, refinement), this aims to reduce the number of animals used in traditional toxicity tests (Tresguerres et al. 2020). Therefore, the aim of this study was to investigate the potential of HSPi in mitigating the toxic effects of BaP in the liver, gill, and kidney cells of Acipenser ruthenus. Specifically, we will assess the effects of HSPi, including Tex-OE and AMG, together with a newly synthesized pirano-piranazole-based inducer of HSPs called SZ, on HSP27, HSP70, and HSP90 proteins expression, liver indicator enzymes, CYP450 and AChE activity, antioxidant and cortisol levels, and immunological responses. By doing so, we hope to provide insights into the mechanisms underlying the protective effects of HSP induction and contribute to the development of effective strategies for mitigating the toxic effects of environmental contaminants on sturgeon species.

Materials and methods

Synthesis of the new compound (SZ)

A detailed explanation of the synthesis of 4,4-(4,1-phenylene)bis(5-amino-3-methyl-4,1-dihydropyrano[3,2-c]pyrazole-6-carbonitrile) based on the supplementary data was provided (Supplementary Fig. 1a, b, and c, respectively) (Shahriyari-Nejad et al. 2020).

In vitro culture of liver, gill, and kidney cells

An Acipenser ruthenus measuring 18 cm and weighing 15.28 g was provided by the International Sturgeon Research Institute. The liver, gill, and kidney tissues were dissected under completely sterile conditions and transferred to separate T-25 cm2 flasks. Each flask was cultured and incubated at 22°C and 95% humidity with essential medium supplemented with culture medium [80% Leibovitz's L-15 medium (L15), 20% fetal bovine serum (FBS), 100 U/ml streptomycin/penicillin (1%), and 100 U/ml amphotericin B (1%) prepared by Sigma‒Aldrich]. After approximately 24 h, the culture medium inside the flask was replaced with fresh complete medium. The cells were examined daily for contamination and growth under a microscope. After 10–15 days, liver, gill, and kidney cells formed a monolayer. Cells adhering to the bottom of the flask were washed twice with phosphate-buffered saline (PBS, pH 7.4). The cells were then treated and lysed with trypsin–EDTA (0.25%; Sigma‒Aldrich). The monolayer cells were then dispersed throughout the flask with trypsin–EDTA. After 3 min, the cells were centrifuged for 5 min at 3000 rpm. The supernatant was discarded, and the cell sediment was transferred to a flask for treatment (Butler 2004; Jamalzadeh et al. 2016).

Determination of BαP IC50 in medium culture and optimal HSPi dose

Treatment with HSP inducers and BaP

5×105 cells/ml were counted from the liver, kidney, and gill and transferred to separate wells of 96-well plates. The cells were incubated at 22°C, 5% CO2, and 95% humidity in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS, 100 U/ml streptomycin/penicillin (1%), and 100 U/ml amphotericin B (1%). After 24 h, the medium was replaced with DMEM containing 1% FBS. Then, the cells were individually treated with different concentrations of BaP (Sigma‒Aldrich): 0, 2.5, 5, 10, 20, 40, and 80 µM, as well as HSPi compounds as follows: Tex-OE (NOP): 0, 50, 100, 200, 400, 800, and 1600 mM, AMG (Sigma‒Aldrich): 0, 1.25, 2.5, 5, 10, 20, 40, and 80 mM, and SZ: 0, 5, 10, 20, 40, 80, and 160 µM for 24 h. A group without treatment was considered a control group. Afterward, cell viability was assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay (Ahmadi et al. 2014; Hasanzadeh et al. 2021). The final volume of each well was 200 µl, and each concentration was repeated three times.

MTT assay

The MTT assay measures cell metabolic activity to determine viability, proliferation, and cytotoxicity. Therefore, to check the toxicity of HSPi compounds on cells, determine their optimal dose, and obtain the IC50 of BαP, an MTT test was used. By reducing the brightly colored tetrazolium salt to its formazan form, a purple‒blue color is produced, whose intensity can be measured spectrophotometrically. At the end of the treatment period, all the wells were empty, and fresh medium without FBS was mixed with 10 µl (10% of final volume) of solution MTT (5 mg/ml, Sigma‒Aldrich), and the plates were incubated at 22 °C, 5% CO2, and 95% humidity for 3–4 h. The final volume of each well was 100 µl, the supernatant was removed, and the formazan crystals formed were solubilized in 100 μl of dimethyl sulfoxide (DMSO; Sigma‒Aldrich) for 30 min. The absorbance at 570 nm was measured using a spectrophotometer, and the results were expressed as a percentage of the control (which is considered 100%) (Grela et al. 2018). Based on the results of the MTT assay, the optimal doses of HSPi were selected: 800 mM NOP (N800), 80 mM AMG (A80), and 80 μM SZ (SZ80) (Supplementary Fig. 2a, b, and c, respectively). In addition, the half-maximum inhibitory concentration (IC50) of BaP values was calculated via probit analysis using POLO-PC software (LeOraSoftware lnc 1987. Berkeley, CA, USA) (Robertson et al. 2017). The IC50 of BaP in medium culture was 30, 32.5, and 31 µM for liver, gill, and kidney cells, respectively. 75% of the IC50 of BaP (B75) was selected for subsequent tests (Supplementary Fig. 2d). The percentage of inhibition and viability was calculated using the following formula (Grela et al. 2018):

$$\mathrm{Percentage\;of\;inhibitory}=[ 1-(\frac{\mathrm{OD }({\text{treatment}})}{\mathrm{OD }({\text{control}})})] \times 100\mathrm{\%}$$
$$\mathrm{Cell\;viability }= [ \frac{\mathrm{OD }({\text{treatment}})}{\mathrm{OD }({\text{control}})}]\times 100\mathrm{\%}$$

BaP concentration in medium and cell samples determination by gas chromatography‒mass spectrometry (GC–MS)

In T-175 cm2 cell culture flasks, a large number of cells were seeded and allowed to become confluent. Control flasks without cells, along with L-15 medium and 75% of the IC50 (B75) of each cell line, were transferred with 3 experimental repeats to determine the amount of adsorption to the plastic well. Additionally, 5 × 105 cells/ml for flasks with cells of the liver, gill, and kidney, complete culture medium [L15 medium, 1% FBS, 100 U/ml streptomycin/penicillin (1%) and 100 U/ml amphotericin B (1%)] and 75% of the IC50 (B75) of each cell line (22.5, 24.5 and 23.5 µM, respectively) were transferred with 3 experimental repeats to determine the amount of adsorption to the plastic well. The wells were incubated at 22°C and 95% humidity for 24 h. The BaP analysis was based on a protocol previously developed and validated by Langan et (Langan et al. 2018). Briefly, to standardize data reported in the literature, cell samples were scraped into glass vials, while aqueous medium samples (10 ml) were transferred from the cell culture bottle to analytical/chromatography glass vials (Sigma‒Aldrich) along with dichloromethane (DCM; 2 ml, Sigma‒Aldrich). Afterward, BaP-d12 (100 ng in 20 μl acetone) was used as a standard. DCM (2 ml) was added, and the cells were extracted in a sonication bath for 20 min. The work continued according to the protocol.

Grouping of treatments

In the grouping of treatments, 5 × 105 cells/ml were counted and transferred to 24-well plates with culture medium [90% DMEM, 10% FBS, 100 U/ml streptomycin/penicillin (1%) and 100 U/ml amphotericin B (1%)]. They were incubated at 22°C, 5% CO2, and 95% humidity. After 24 h, the culture medium was replaced (with 1% FBS), and the cells were divided into four groups (Supplementary Fig. 3a): 1) the control (without any BaP or HSPi), 2) the cells that received different concentrations of HSPi (N800, A80, and SZ80), 3) the cells subjected to only BaP (B75), and 4) the cells treated with HSPi that received BaP (HSPi + B75). Each group was repeated 3 times, and after 24 h, the cells were collected from the wells and used for the next tests. The final volume of each well was 500 µl. In all cases, aseptic conditions were followed. The total protein of each treatment group (5 × 105 cells/ml) was determined using Bradford (Bradford 1976), with bovine plasma albumin (Sigma‒Aldrich) as a standard (Supplementary Fig. 4).

Western blotting analysis

Western blotting was used to analyze the expression patterns of HSP27, HSP70, and HSP90 as described by Werner et al. (Werner et al. 2007). Briefly, sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) was performed on equal amounts of protein (25 μg) from each sample (12% polyacrylamide gel and 5% stacking gel). The gels were then electroblotted onto a total polyvinylidene fluoride transfer membrane (Millipore, Bedford, Massachusetts). A primary antibody was used to assay HSP27, HSP70 and HSP90 immunoblots (sc-13132 (Santa Cruz Biotechnology, INC), H5147, and H1775 from Sigma‒Aldrich). For ß-actin, which is a housekeeping protein (control), monoclonal anti-ß-actin (A3854) and anti-mouse IgG-peroxidase conjugate (A2304, Sigma‒Aldrich) were utilized as primary and secondary antibodies, respectively. 3,3′-Diaminobenzidine (DAB; Sigma‒Aldrich, D-7304) and H2O2 were used as substrates.

Note: Gill cells were analyzed using western blots, and the enzyme assay was not performed on these cells.

Assay of CYP450 activity

CYP450 activities in liver cells were determined using a Fish Cytochrome P450 1A1 (CYP1A1) ELISA Kit (Fish ELISA Kit, EASTBIOPHARM CO, USA) (Focardi et al. 2000; Yildirim et al. 2014). The treated cells were suspended in 50 µl chilled cell lysis buffer (Section 2.3.4) and centrifuged for 20 min at 12,000 rpm and 4°C. All steps were completed according to the kit instructions, and optical density (OD) was determined at 450 nm using a 3-replicate ELISA reader.

Antioxidant assay in liver cells

The glutathione-s-transferase (GST) activity was determined by increasing the absorbance at 340 nm with 1 mM 1-chloro-2,4-dinitrobenzene (CDNB, Sigma‒Aldrich) and 1 mM GSH evaluated in 100 mM Na-phosphate buffer pH 6.5 (Pacini et al. 2013).

The activity of glutathione peroxidase (GPx) was measured at 340 nm following the oxidation of NADPH in a buffer containing 100 mM Na-phosphate with pH 7.5, 1 mM EDTA, 0.12 mM NADPH, 2 mM GSH, 1 mM NaN3, and 1 U glutathione reductase (GR) (ZellBio GmbH, Germany).

Similar to GPx, the total antioxidant capacity (TAC) levels were assessed using an ELISA kit (ZellBio GmbH, Germany) at 490 nm (Q. Li et al. 2017).

Immune responses in liver cells

Quantitative levels of complement component 3 (C3) were determined using an ELISA sandwich (enzyme-linked immunosorbent assay) with a fish ELISA kit (Hangzhou East Biopharm Co. Ltd) (Baharloei et al. 2020). An antibody-enzyme monoclonal well was previously coated with C3 fish monoclonal antibody to form an immune complex when biotin-labeled C3 antibody was mixed with streptavidin–horseradish peroxidase at 37°C. To remove the unmixed enzyme, the plate was washed. The liquid color changed to yellow upon adding sulfuric acid. The OD value was measured with a microplate reader at 450 nm, and C3 concentrations were expressed.

Immunoglobulin M (IgM) was measured using an ELISA quantification kit (Hangzhou Eastbiopharm Co. Ltd) (Valipour et al. 2018). IgM standards and supernatant samples were analyzed manually. After all the necessary steps were completed, the OD at 450 nm was determined within 15 min for both IgM standards and supernatant samples. The measurements were repeated three times.

Lysozyme activity (LYZ) was assessed using a turbidimetric method described by Kim and et al. (Kim et al. 2019). A substrate of Micrococcus lysodeikticus (Sigma‒Aldrich) was used at a concentration of 0.2 mg/ml in 0.05 M phosphate buffer, pH 6.6. A standard curve was constructed using lyophilized chicken egg white LYZ, and the rate of change in turbidity was measured at 530 nm. The results correspond to chicken egg white LYZ activity.

Measurement of cortisol levels and liver indicator enzymes

The enzymatic activity of aspartate aminotransferase (AST, EC 2.6.1.1), alanine aminotransferase (ALT, EC 2.6.1.2), alkaline phosphatase (ALP, EC 3.1.3.1), and lactate dehydrogenase (LDH, EC 1.1.1.27) was studied in liver and kidney cells according to the strategies recommended by the World Federation of Clinical Chemistry (IFCC) and the protocol provided with the kit (MyBioSource, USA) without pyridoxal phosphate. This approach is proposed for fish enzyme analysis (Barulin 2015).

Cortisol levels were quantified in liver cells using a commercial enzyme-linked immunosorbent assay (ELISA) kit (Nanjing Jiancheng Institute, Nanjing, China) according to the manufacturer's recommended instructions. The color changes were detected spectrophotometrically at 450 nm (Long et al. 2019).

AChE-specific activity

AChE-specific activity in liver and kidney cells was determined by Ellman (Ellman et al. 1961). Lysis buffer (phosphate buffer containing Triton X-100: 0.1 M at pH 7.2) was used to treat the cells, followed by centrifugation (at 12000 rpm for 20 min at 4 °C), and the supernatant was obtained. The reaction was started with the addition of the substrate acetylthiocholine (AcSCh, Sigma‒Aldrich). A microplate reader was used to measure the hydrolysis and formation of the dianion of 5,5′-dithio-bis-(2-nitrobenzoesäure) (DTNB, Sigma‒Aldrich) within 415 nm for 5 min (at intervals of 1 min).

Statistical analysis

Statistical analysis was conducted using SPSS/PC + 23 (SPSS Inc.) and GraphPad Prism 8 software. All experiments were repeated three times, and numerical data are presented as the mean ± SEM. Prior to analysis, data normality was confirmed using the one-sample Kolmogorov‒Smirnov test. To determine significant differences between groups, a one-way analysis of variance (ANOVA) followed by Duncan's test for multiple comparisons was performed. Principal component analysis (PCA) was employed to assess the main components among the studied parameters and different treatments for BaP stress. The relationship between biomarkers and fish cells was evaluated using Pearson correlation analysis. A significance level of p < 0.0001 was adopted.

Results

Cell viability

Viability evaluation of cells undertreated with HSPi and BaP

Liver, gill, and kidney cells were separately exposed to different concentrations of HSPi compounds, including NOP, AMG, and SZ, as well as BaP pollutants for 24 h (Supplementary Figs. 2a, b, c, and e, respectively; p < 0.0001). Among the HSPi compounds, AMG (at a concentration of 80 mM: 153.66% in liver, 151.56% in gills, and 152.33% in kidney) and SZ (at a concentration of 160 µM: 137.33% in liver, 144.48% in gills, and 142.66% in kidney) showed the best survival rates in all three cell lines compared to the control group (100%). In contrast, higher NOP concentrations (at 1600 mM: 80.33% in liver, 80.66% in gills, and 78.29% in kidney) resulted in lower survival rates (p < 0.0001). Unlike HSPi, liver, gill, and kidney cells treated with various BaP concentrations showed a significant decrease in viability with increasing doses (p < 0.0001). There was a significant difference in the viability of cells treated with 80 µM BaP (23.5% in liver, 22.5% in gills, and 22.4% in kidney) compared to the control (p < 0.0001). The IC50 of the BaP pollutant was estimated to be 30, 32.5, and 31 µM for liver, gill, and kidney cells, respectively. For subsequent testing, 75% of the IC50 (B75) was selected. Furthermore, the optimal dose of HSPi was determined using cell survival results: for NOP, 800 mM (N800); for AMG, 80 mM (A80); and for SZ, 80 µM (SZ80).

Overview of cell viability at selective doses (%)

We grouped treatments together, as shown in Supplementary Fig. 3a, and assessed their survival rates (Supplementary Fig. 3b). The levels of cell survival for the chosen dose treatments for liver, gill, and kidney cells differed. In all three treated cell tissues, B75 (in the liver, 69.64%; in the gills, 70.95%; and in the kidney, 71.29%) had a reducing effect on cell viability (%) compared to the control (100%; p < 0.0001). N800 had the lowest viability among all HSPi compounds (liver: 89.76%, gill: 90.61%, and kidney: 89.55%). Compared to the B75 group, the HSPi + B75 groups, including SZ80 + B75 (in liver 99.73%, in gill 97.4%, and kidney 98%), A80 + B75 (in liver 107.5%, in gill 105.12%, and kidney 103.72%), and treatment with N800 + B75 (liver: 94.93%, gill: 100%, and kidney: 103%), each had significantly increased cell survival (p < 0.0001).

The medium cultured without cells (control) and the medium with three cell types (liver, gill, and kidney) were treated with 75% of the IC50 (B75) of each cell line (22.5, 24.5, and 23.5 µM, respectively) for 24 h (Supplementary Fig. 3c). A significant difference in BaP concentration was observed in the culture medium without and with cells. After 24 h, the cells absorbed 12% to 18% BaP from the medium.

HSP response to the BaP pollutant

HSP27

HSP27 protein expression varied significantly in liver, gill, and kidney cells (Fig. 1; p < 0.0001). In the liver (Fig. 1a and d), compared to the control group, the lowest and highest levels of HSP27 expression were observed in treatments with B75 (0.055-fold) and A80 (1.344-fold), respectively (p < 0.0001). SZ80 had the lowest HSP27 expression levels among all HSPi compounds, indicating its weaker effect on HSP27 regulation (0.144-fold). Compared to the control groups, treatment with A80 + B75 (0.643-fold) and N800 + B75 (0.161-fold) significantly decreased HSP27 expression but increased expression compared to the B75 group (p < 0.0001). In the gills (Fig. 1b and d), the B75 group (3.261-fold) had a more significant effect on HSP27 expression than the control group. Treatment with N800 also showed maximal HSP27 expression (4.483-fold; p < 0.0001). Similar to liver cells, SZ80 (1.155-fold) exhibited minimal protein expression levels in gill cells among all HSPi compounds (p < 0.0001). Moreover, compared to the control group, A80 + B75 (0.928-fold) and SZ80 + B75 (0.255-fold) HSP27 expression was significantly reduced (p < 0.0001). In kidney cells (Fig. 1c and d), SZ80 + B75 (0.033-fold) and N800 (1.155-fold) cells expressed less and more HSP27 than controls, respectively. The B75 group (0.301-fold) had less effect on HSP27 expression than the control group. Moreover, N800 + B75 (0.569-fold) significantly increased HSP27 expression compared to SZ80 + B75 (0.033-fold) and A80 + B75 (0.440-fold; p < 0.0001).

Fig. 1
figure 1

HSP27 protein relative expression analysis through western blotting. a) Liver, b) gill, and c) kidney cells. d) HSP27 protein levels are shown as fold of control values (mean ± SEM), and within each column, different letters indicate significantly different groups using Duncan's test (a, b, c, and…; P < 0.0001). 8 groups were considered: 1) control cells without treatment (Cr), HSPi groups containing 2) 80 mM amygdalin (A80), 3) 80 µM SZ (SZ80), 4) 800 mM Nopal Endurance (N800), 5) stress group just 75% IC50 BaP (B75), HSPi + stress groups containing HSPi + B75: 6) A80 + B75, 7) SZ80 + B75, and 8) N800 + B75

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HSP70

Due to BaP and HSPi treatment, HSP70 protein expression fluctuated in liver, gill, and kidney cells (Fig. 2; p < 0.0001). In the liver (Fig. 2a and d), B75 (0.461-fold) had a decreasing effect on HSP70 protein expression compared to the control (p < 0.0001). SZ80 (0.351-fold) decreased HSP70 expression, while N800 (1.236-fold) increased it. Compared to the B75 group, the A80 + B75 group (0.778-fold) showed a slight increase in HSP70 expression (p < 0.0001). In the gill (Fig. 2b and d), BaP (0.416-fold) had less effect on HSP70 protein expression than the control. Treatments N800 (2.172-fold) and SZ80 (0.064-fold) expressed the most and the least HSP70, respectively. N800 + B75 (0.778-fold) slightly improved the expression of HSP70 (p < 0.0001). In the kidney (Fig. 2c and d), HSP70 expression was reduced in SZ80 + B75 (0.768-fold) and increased in N800 (5.573-fold). Among all HSPi compounds, SZ80 had the lowest protein expression levels. In addition, the change in B75 (0.822-fold) was less than that of the control. N800 + B75 (3.740-fold) significantly increased HSP70 expression compared to SZ80 + B75 (0.768-fold) and A80 + B75 (1.280-fold; p < 0.0001).

Fig. 2
figure 2

Analyzing HSP70 protein relative expression by western blotting. a) Liver, b) gill, and c) kidney cells. d) HSP70 protein levels are shown as fold of control values (mean ± SEM), and within each column, different letters indicate significantly different groups using Duncan's test (a, b, c, and…; P < 0.0001). 8 groups were considered: 1) control cells without treatment (Cr), HSPi groups containing 2) 80 mM amygdalin (A80), 3) 80 µM SZ (SZ80), 4) 800 mM Nopal Endurance (N800), 5) stress group just 75% IC50 BaP (B75), HSPi + stress groups containing HSPi + B75: 6) A80 + B75, 7) SZ80 + B75, and 8) N800 + B75

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HSP90

After BaP and HSPi treatment, HSP90 protein expression changed in liver, gill, and kidney cells (Fig. 3; p < 0.0001). In the liver (Fig. 3a and d), the highest HSP90 expression was observed in N800 (1.648-fold), and the lowest expression was observed in B75 (0.129-fold). Compared to B75, N800 + B75 showed a significant increase of 0.758-fold in HSP90 expression (p < 0.0001). In the gills (Fig. 3b and d), treatment groups A80 (2.448-fold) and B75 (0.158-fold) had higher and lower HSP90 expression, respectively. Moreover, among all HSPi compounds, SZ80 (0.286-fold) showed the lowest protein expression. A80 + B75 (1.546-fold) and N800 + B75 (1.730-fold) treatments notably increased the expression of HSP90 (p < 0.0001). In the kidney (Fig. 3c and d), HSP90 expression was lowest in treatment B75 (0.120-fold). N800 (1.872-fold) exhibited the highest protein expression range among all HSPi compounds. N800 + B75 (0.746-fold) significantly increased HSP90 expression compared to A80 + B75 and SZ80 + B75 (0.147-fold and 0.132-fold, respectively; p < 0.0001).

Fig. 3
figure 3

An analysis of the relative expression of HSP90 protein by western blotting. a) Liver, b) gill, and c) kidney cells. d) Each column shows fold of control values (mean ± SEM), and different letters indicate different groups using Duncan's test (a, b, c, and…; P < 0.0001). 8 groups were considered: 1) control cells without treatment (Cr), HSPi groups containing 2) 80 mM amygdalin (A80), 3) 80 µM SZ (SZ80), 4) 800 mM Nopal Endurance (N800), 5) stress group just 75% IC50 BaP (B75), HSPi + stress groups containing HSPi + B75: 6) A80 + B75, 7) SZ80 + B75, and 8) N800 + B75

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Note: The original photos of the western blot gel are available in the Supplementary Figs. 5, 6, and 7.

CYP450 activity

To assess liver cell integrity, we determined the effect of BaP and HSPi treatment on CYP450 activity (Fig. 4; p < 0.0001). The B75 group exhibited the highest CYP450 activity, while the control groups showed the lowest levels. We observed that CYP450 activity was similar for all HSPi compounds in N800 and A80. Administration of BaP to HSPi-treated cells (HSPi + B75) significantly attenuated the increase in CYP450 activity (p < 0.0001).

Fig. 4
figure 4

Evaluation of CYP450 activity in A. ruthenus liver cells (mean ± SEM). BaP treatment drastically increased CYP450 activity. However, treatment with HSPis for 24 h decreased the activity of these proteins. Within each column, different letters indicate significantly different groups using Duncan's test (a, b, c, and…; P < 0.0001)

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AChE-specific activity

In terms of AChE-specific activity, both liver and kidney cells exhibited similar patterns (Fig. 5; p < 0.0001). B75 had an inhibitory effect on AChE activity, with the lowest values observed in the liver (9.80 nmol/min/mg protein) and kidney (8.32 nmol/min/mg protein) compared to the control group (liver 19.70 and kidney 21.10 nmol/min/mg protein). All HSPi compounds exhibited higher activity than the B75 group. Furthermore, HSPi + B75 significantly increased AChE enzyme activity in both liver and kidney cells compared to the BaP group (p < 0.0001).

Fig. 5
figure 5

Assessing AChE activity in A. ruthenus liver and kidney cells. BaP significantly decreases AChE activity, and HSPis, particularly SZ, can reverse this effect. The vertical bar denotes a standard error (mean ± SEM), and within each column in the chart, different letters indicate significantly different groups using Duncan's test (a, b, c, and…; P < 0.0001)

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Antioxidant activity

By measuring GST, GPx, and TAC, the antioxidant activity of liver cells was assessed (Table 1; p < 0.0001). All three enzymes showed similar activity levels. Treatment with HSPi led to a significant increase in hepatic GST, GPx, and TAC levels compared to those in the control group. The control group showed the lowest degree and most antioxidant parameters (GST: 0.245 U/g, GPx: 1.842 U/g, and TAC: 6.850 μmol/g), followed by the N800 group (GST: 0.388 U/g, GPx: 2.882 U/g, and TAC: 11.38 μmol/g). Furthermore, the B75 group had a greater effect on antioxidant activity than the control group (GST: 0.313 U/g, GPx: 2.186 U/g, and TAC: 7.715 μmol/g). In general, the HSPi + B75 groups performed better than the control and B75 groups in terms of the enzymes mentioned (p < 0.0001).

Table 1 Antioxidant activity in liver cells of sterlet sturgeon (mean ± SEM; p < 0.0001)
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Immune responses

In terms of immune responses, liver cells exposed to different substances were examined for IgM, C3, and LYZ activities (Table 2; p < 0.0001). The activity levels of C3 were apparently increased in the control (1.55 mg/g) and SZ80 (1.52 mg/g) treatments, while a decrease in activity was clearly observed in the A80 + B75 (1.00 mg/g) treatment. Compared to the control, the B75 group had a lower C3 level (1.41 mg/g) while it was slightly increased in the N800 + B75 (1.49 mg/g) group (p < 0.0001). IgM levels in the B75 group (2.58 mg/g) were higher than those in the control group (2.49 mg/g), and the HSPi groups (A80: 2.41, SZ80: 2.51, and N800: 2.21 mg/g) were less active than the B75 group. Treatments with N800 + B75 (2.66 mg/g) and SZ80 + B75 (2.54 mg/g) showed the highest levels of IgM (p < 0.0001). LYZ enzyme changes were similar in the B75 and control groups (0.155 U/g/min). Overall enzyme activity was reduced in the HSPi and HSPi + B75 treatment groups (p < 0.0001).

Table 2 Immune responses in liver cells in sterlet sturgeon (mean ± SEM; p < 0.0001)
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Liver indicator parameters and cortisol levels

The liver enzymes, such as AST, ALT, ALP, and LDH, and cortisol levels, varied in liver cells treated with BaP and HSPi (Table 3; p < 0.0001). The B75 (0.078 ng/g) and HSPi groups (A80: 0.076, SZ80: 0.085, and N800: 0.080 ng/g) showed an increasing effect on cortisol levels compared to the control (0.060 ng/g; p < 0.0001). In the HSPi + B75 groups, SZ80 + B75 (0.076 ng/g protein) and N800 + B75 (0.076 ng/g protein) showed similar changes, unlike treatment A80 + B75, which decreased (0.033 ng/g protein; p < 0.0001). The pattern of changes in AST, ALT, ALP, and LDH enzymes was similar. Compared to the control group, the B75 group significantly increased all enzyme activity values (p < 0.0001). Based on the results obtained for all HSPi compounds, it was estimated that SZ80 treatment had the highest levels of enzymes (excluding LDH) (p < 0.0001). Compared to the B75 group, the groups exposed to inducers first and then to BaP (HSPi + BaP) showed a significant decrease in enzyme activity (p < 0.0001).

Table 3 Liver indicator enzymes in liver cells of sterlet sturgeon (mean ± SEM; p < 0.0001)
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In terms of AST, ALT, ALP, and LDH levels, kidney cells exhibited relatively similar results as liver cells (Table 4; p < 0.0001). ALP and LDH showed similar patterns to AST and ALT. Comparing the B75 group with the control group, the B75 group demonstrated a significant increase in enzyme activity (p < 0.0001). For the enzymes mentioned, SZ80 had the highest activity of all HSPi compounds. Compared to B75, HSPi + B75 treatment showed lower activity in AST, ALT, ALP, and LDH enzymes (p < 0.0001).

Table 4 Changing liver indicator enzymes in sterlet sturgeon kidney cells (mean ± SEM; p < 0.0001)
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Assessing PCA

We conducted PCA on 32 variables and 24 treatments, which revealed that the first six principal components accounted for 45.21%, 19.68%, 12.09%, 9.82%, 4.33%, and 3.63% of the changes, respectively (Table 5). We used variables with a score higher than 0.6 to interpret the components (Supplementary Fig. 8, Table 1 and 2). The variables measured in BaP stress played a significant role in determining the relationship between the treatments and the tested variables in the first to third components (Fig. 6). Finally, we performed a Pearson’s correlation analysis on all measured parameters (Supplementary Table 3).

Table 5 Initial eigenvalues including total, percentage of variance, and cumulative percentage of variance related to all six components
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Fig. 6
figure 6

Principal component analysis (PCA) for BaP stress. Six components captured 94.80% of the total variation. A significant relationship was found between the treatments and all variables measured under BaP stress in the first three components. The abbreviations are L: liver, G: gill, K: kidney, LV: liver viability, GV: gill viability, and KV: kidney viability

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Discussion

Sturgeons have been cultivated artificially in the Caspian Sea due to extinction conditions in recent years (Prokopchuk et al. 2016). The Caspian Sea has been extensively polluted by oil industries, which are the main source of PAHs in this ecosystem (Lattuada et al. 2019).

BaP upregulates HSP proteins, particularly HSP70 and HSP90, by causing cellular stress (Bakhtyar and Gagnon 2011). Additionally, many studies have shown that AChE enzyme activity is inhibited due to BaP toxicity (Olivares-Rubio and Espinosa-Aguirre 2021). Oxidative stress is considered one of BaP's major causes. The induction of antioxidant (AOX) biomarkers as an indication of oxidative stress has been documented in a number of fish species after exposure to BaP (Woo and Chung 2020). Despite a wide range of toxicological studies describing the effects of BaP on fish, little is known about the metabolic mechanisms responsible for these effects. Previous studies have shown that BaP is a serious environmental pollutant that accumulates in invertebrates and poses toxicity risks to fish (Esmaeilbeigi et al. 2021; Zena et al. 2015). The present study focused on assessing the effects of BaP on various biological parameters, including HSP27, HSP70, and HSP90 proteins expression. It also evaluated liver indicator enzymes, AChE, and CYP450 activities, antioxidant and cortisol levels, and immunological responses. Further, the authors of this study assessed the impact of Tex-OE and AMG, and newly synthesized pirano-piranazole-based SZ as HSP inducers on mitigating BaP toxicity.

Acipenser ruthenus was used as an ecological model to assess the viability of liver, kidney, and gill cells in the presence of HSP inducers and BaP. Although sturgeon fish are sensitive to PAHs, including BaP, they have not been extensively studied. We hypothesized that inducing HSPs in sterlet cell lines could increase protection against BaP in isolated sterlet cells from the liver, gills, and kidney. To assess the potential harm caused by these compounds, toxicity tests were conducted on zooplankton and cyanobacteria. According to Ikenaka et al. (2013), Daphnia magna and Ceriodaphnia reticulata have different LC50 values for BaP. For Ceriodaphnia reticulata, the LC50 value of BaP was 4.3 g/l, and for Daphnia magna, it was 4.7 g/l (Ikenaka et al. 2013). In the present study, we first used the MTT assay and found that BaP can considerably reduce the viability of sterlet cells (IC50: 30, 32.5, and 31 µM for cells from the liver, gill, and kidney, respectively) (Supplementary Fig. 2). Next, samples of medium and cells were analyzed using GC‒MS to determine BaP concentrations (Supplementary Fig. 3c). Different fish species may exhibit resistance to pesticides and water quality variations (Jiang 2022).

Several studies have reviewed the role of HSPs in cells' protective mechanisms against chemicals and have indicated that HSPs could serve as early warning indicators of environmental pollution (Liu et al. 2015; Mrdaković et al. 2016). As a result of the upregulation of HSP70, it appears that HSPs are responsible for breaking down or eliminating BaP from an organism, thereby reducing its toxicity. According to Gao et al. (Gao et al. 2022), acute BaP toxicity enhances HSP70 and HSP90 induction in the clam Ruditapes philippinarum. Therefore, treatment with BaP changed the expression of HSP27, HSP70, and HSP90 in all three cell lines (Fig. 1, 2, and 3, respectively). BaP could also greatly reduce AChE activity and enhance cell detoxification by upregulating CYP450 activation (Fig. 5 and Fig. 4, respectively). Finally, BaP boosted GST, TAC, and GPx enzyme oxidative activity (Table 1). Additionally, BaP increased IgM immune parameters and decreased C3 levels (Table 2). It could also increase the cortisol levels and liver indicator parameters, including AST, ALT, ALP, and LDH, in liver and kidney cell lines (Table 3 and 4, respectively).

Several studies have reported that Tex-OE increases HSP70 expression in fish and shellfish (Baruah et al. 2014; Vahdatiraad et al. 2023a; Vahdatiraad et al. 2023b). However, there are no studies related to the effect of AMG and pirano piranazole-derived compounds on HSP expression in sturgeon fish. Following our initial findings regarding BaP, we encountered significant challenges: 1) HSP inducers can cause the same alterations as BaP without reducing cell viability and increasing cell death. 2) The combination of HSP with BaP can restore conditions back to normal. Given the crucial role of HSP chaperones in safeguarding cells against stress and their connection with BaP, we explored the potential of using HSP inducers to counteract the effects of BaP on A. ruthenus cell lines. To test this hypothesis, we employed NOP, AMG, and a pirano piranazole-derived compound called SZ as HSPi (Supplementary Fig. 1). First, the viability of cells after treatment with the mentioned compounds was examined to further investigate this challenge. According to the MTT assay (Supplementary Figs. 2 and 3), NOP decreased cell viability, while AMG and SZ increased cell viability.

Fish farming emphasizes the first aspect of conservation. Raising endangered fish on special farms and releasing them into freshwater after they mature is one way to conserve their population. However, a disadvantage of this approach is that the fish are raised in optimal conditions inside the farms, but they will be exposed to different types of water pollution after release. As a result, it is possible that the first aspect of HSPis use can alleviate this problem. However, the second aspect of the hypothesis, in which BaP was combined with the HSPis in the tests, is critical to nullifying BaP toxicity.

24-h exposure to BaP was associated with BaP influx into cells, as well as a decrease in BaP in the medium, indicating active enterocytic transport in this model, although identification of the metabolites produced was not feasible (Supplementary Fig. 3c). We hypothesize that the cells eject BaP back into the medium in a nontoxic form as a means to prevent resorption of this compound, a process common in animal systems. It seems that liver cells, due to their antioxidant and detoxification systems, absorb less BaP from the environment than gills and kidneys.

At HSP levels, NOP and AMG inducers performed better in all three cell lines tested. Additionally, SZ increased HSP90 expression in liver cells to some extent (Fig. 1, Fig. 2, and Fig. 3). The combination of SZ with BaP produced different results than the control group.

Previous researchers demonstrated a clear correlation between CYP450 gene activation and HSP expression under stressful conditions (Li et al. 2016; Sang et al. 2012). BaP causes the activation and increase of the CYP450 enzyme (Fig. 4). Therefore, in addition to HSP upregulation, CYP450 activation by BaP is observed. Current studies suggest that HSPs and CYP450 have a chaperone/client relationship. HSPs protect CYP450 activity in stressful situations. HSPi combined with BaP (HSPi + B75) could reduce CYP450 activity compared to BaP alone (B75).

Ecotoxicological studies have shown that pesticides, PAHs, carbamates, and metals decrease AChE activity in fish (Oliveira et al. 2015; Taheri et al. 2022). These data highlight the importance of investigating the effects of organic pollutants on aquatic organisms such as fish (Fig. 5). HSPi combined with BaP (HSPi + B75) could increase AChE activity compared to BaP alone (B75). HSP inducers can reduce BaP damage in fish and increase the activity of the referred enzyme (especially SZ).

By generating reactive oxygen species (ROS) and inducing oxidative stress, PAHs adversely affect aquatic organisms (Soltani et al. 2019). Therefore, strengthening fish antioxidant activity and immune responses is considered an appropriate way to improve the survival rate and overall health against water pollution (Palanikumar et al. 2012; Tarazi et al. 2021). ROS can be minimized by using antioxidant enzymes such as GST, GPx, and TAC, which neutralize or repair their harmful effects. Depending on the HSPi, antioxidant parameters were increased individually or in combination with BaP (HSPi + BaP; Table 1). Overall, our findings suggest that HSP-inducing compounds can help control ROS and enhance antioxidant defense mechanisms in sterlet sturgeon cells facing environmental stress. Aquatic organisms may benefit from this approach by mitigating the harmful effects of BaP exposure and other environmental stressors.

It is widely recognized that PAHs and other organic chemicals have a negative impact on fish immune function (Rehberger et al. 2017; Shirmohammadi et al. 2018). Studies have shown that HSP-inducing compounds increase plasma concentrations of IgM, C3, and LYZ in sturgeon (Baharloei et al. 2020; Vahdatiraad et al. 2023a). The present study showed that NOP, to some extent, enhanced immunological responses in liver cells exposed to BaP (N800 + B75). However, the other two HSPis (AMG and SZ) could not improve the immune system in the presence of BaP (Table 2).

In the present study, liver cells that received HSPi and BaP (B75) showed increased cortisol levels compared to those in the HSPi + BaP groups. The decrease in cortisol levels in the HSPi + BaP groups may be attributed to the relationship between cortisol and HSP synthesis (Celi et al. 2012). Several signaling pathways are involved in HSP production, the most influential being the hypothalamus-pituitary-interrenal (HPI) axis, which controls cortisol production. Reduced HPI axis function results in a negative feedback loop on cortisol secretion (Vahdatiraad et al. 2023a). In the cytosol, HSP70 and HSP90 form a stable heterocomplex with the glucocorticoid receptor (GR), inhibiting cortisol secretion and synthesis (Celi et al. 2012). Due to their chaperone/client relationship with cortisol, HSPs play a crucial role in regulating cortisol activity during stressful situations. This idea is supported by the finding of reduced cortisol activity in the HSPi + BaP group compared with the BaP and HSPi groups (Table 3). Stress levels in fish can be determined by liver indicator enzymes (AST, ALT, ALP, and LDH) (Banaee et al. 2013). Finally, AST, ALT, ALP, and LDH can be used to determine stress levels in fish. As mentioned, BaP increased these parameters. Treatment of liver and kidney cells with HSPi + BaP showed a significant decrease in enzyme agents (Table 3 and Table 4, respectively). HSPis may be able to minimize BaP damage.

Based on PCA and Pearson correlation, HSP expression in different cells was found to be related to most of the parameters studied (Table 5, Supplementary Table 1 to 3). This highlights the importance of this family in different biological states. Using PCA, we identified the most effective treatment for BaP stress. Based on this analysis, we found that HSPs expression is directly related to liver indicator enzymes, CYP450, and AChE enzymes, and antioxidant parameters (Fig. 6). The use of AMG and NOP alongside BaP can most effectively return the physiological state back to baseline.

Conclusions

In the present study, we investigated the moderating effects of HSP inducers on the toxicity caused by BaP pollutants in isolated cells of Acipenser ruthenus sturgeon. Our findings suggest that HSP inducers have the potential to positively modify HSPs expression, liver indicator enzymes, and antioxidant activity while maintaining AChE and CYP450 activity in A. ruthenus against BaP, thereby increasing cell survival in vitro. In conclusion, HSP inducers may be a powerful and reliable way to increase A. ruthenus resistance prior to BaP exposure and reverse the harmful effects of BaP. However, further research is needed to fully understand the underlying mechanisms and assess the long-term effects and potential applications of HSP inducers under environmental conditions.