Effects of copper nanoparticles on oxidative stress genes and their enzyme activities in common carp (Cyprinus carpio)

Abstract Copper nanoparticles (Cu-NPs) are becoming increasingly prevalent in the environment due to their wide range of applications, posing potential threats to living organisms. Negative effects of Cu-NP exposure have been confirmed in many fish species, and they include disorders in the expression of oxidative stress genes and the activity of enzymes they encode. Common carp, known for its sensitivity to nanopollutants in water, serves as a valuable model organism for nanoparticle toxicity assessment. This study investigated the effects of specific Cu-NPs – copper nanopowder (Cu-NPs), colloidal copper nanoparticles (Cu), and copper oxide nanoparticles (CuO-NPs) – on gene expression and enzyme activity (GPX, CYP1A, HSP70, SOD, CAT) in C. carpio hatchlings, utilizing molecular biology tools and biochemical analyses. Results showed that Cu increased the expression of the hsp70 gene, Cu-NPs elevated the expression of the cyp1a and hsp70 genes, and CuO increased cyp1a expression. Conversely, sod and cat exhibited reduced expression across all samples (Cu, Cu-NPs, CuO). All Cu forms induced significant ROS accumulation and notable alterations in oxidative stress biomarkers (SOD, CAT, GPX).


Introduction
In the past decade, a wide array of nanomaterials (NMs) has gained widespread global use.Recently, copper, copper-based nanoparticles (Cu-NPs), and copper oxide-based NPs (CuO-NP) have found application areas across diverse sectors, including industry, construction (Gupta & Siddique 2020), electrical equipment (Mitrea et al. 2016), organic synthesis catalysts (Barot et al. 2016;Lennox et al. 2016), antimicrobial agents (Dugal & Mascarenhas 2015), agriculture (Kalatehjari et al. 2015;Montes et al. 2016), transportation and drug delivery (Woźniak-Budych et al. 2016), paint components or water management (Ma et al. 2016;Ighalo et al. 2021).As nanotechnology and Cunanotech-based applications continue to develop, particularly in wastewater treatment, concerns have arisen regarding their potential ecotoxicity to both human health and aquatic organisms.When released into water through various routes in the form of aggregates, a small particles or ions, these nanoparticles can accumulate in aquatic organisms (Hanna et al. 2014;Conway et al. 2015).Copper nanoparticles (Cu-NP) have been shown to induce oxidative stress in fish, leading to tissue and organ damage (e.g., gills and liver), DNA damage, increased mortality, changes in gene expression (including those related to oxidative stress) and alterations in enzyme activities (Aghamirkarimi et al. 2017;Saddick et al. 2017;Yalsuyi & Vajargah 2017).Elevated mortality rates, particularly among juveniles, pose significant challenges for fish farming, resulting in substantial economic losses.One such vulnerable organism is the common carp (Cyprinus carpio), whose production exceeded 4189.5 kilotons in 2018 (FAO 2020).C. carpio, distributed mainly in Asian and European countries, holds significance in freshwater aquaculture due to its ease of rearing, low production cost, and high nutritional value of its muscle tissue (Rahman 2015).Furthermore, C. carpio serves as a crucial freshwater animal model and is extensively studied in ecotoxicological research owing to its heightened sensitivity to toxic substances (Bongers et al. 1998;Kakakhel et al. 2021).Histopathological changes in the gills of C. carpio exposed to Cu-NPs have been observed (Forouhar Vajargah et al. 2018;Noureen et al. 2021), and the presence of Cu-NPs in the organism can lead to metabolic disorders (Mazandarani & Hoseini 2017) and alterations in enzymatic activity (Liu et al. 2017).Fish larvae, being highly susceptible to the toxic effects of nanoparticles, are suggested to be more adversely affected than other life stages (Johari et al. 2013;Ma & Diamond 2013).Hence, investigating the impact of released Cu-NPs on the molecular and biochemical parameters of C. carpio, especially its larvae, is imperative.
Enzymes such as glutathione peroxidase (GPX), catalase (CAT) and superoxide dismutase (SOD) serve as the primary defense against oxidative stress by orchestrating a coordinated cellular response (Margis et al. 2008).SOD catalyzes the dismutation of two superoxide anion radicals into hydrogen peroxide (H 2 O 2 ) and oxygen.Subsequently, catalase facilitates the reduction of H 2 O 2 to water and oxygen (Ighodaro & Akinloye 2018).Extending beyond this primary defense, cytochromes P450, notably cytochrome P450 1A2, actively engage in xenobiotic metabolism, augmenting the cellular defense against external stressors (Široká & Drastichova 2004).The evaluation of both CYP1A2 expression and activity serves as indispensable biomarkers for the sensitive detection of environmental pollutants (Goksøyr 1995).Concurrently, cellular defense encompasses genetic regulation, with the upregulation of heat shock genes (hsp) in response to diverse stressors.This heightened expression strengthens cellular defense mechanisms against various stressors, including heat, reactive oxygen species (ROS), and toxic compounds (Schlesinger 1990;Abravaya et al. 1992).Collectively, these genes and the enzymes they encode form an intricate network, playing crucial roles in the cellular response to reactive oxygen species (ROS) and, consequently, oxidative stress.This comprehensive defense strategy extends to challenges induced not only by inherent oxidative stress but also by the presence of nanoparticles within the cellular environment.
Numerous studies document the deleterious impact of copper nanoparticles on tissues and the bioaccumulation in fish (Song et al. 2015;Shahzad et al. 2018).However, a notable gap in existing knowledge pertains to the absence of data concerning alterations in oxidative stress-related gene expression and enzymatic activity.Therefore, the primary objective of this study was to investigate the effect of copper nanoparticles in colloidal form (Cu), copper nanopowder (Cu-NPs) and copper oxide (CuO-NPs) on both the expression of genes related to oxidative stress and the activity of enzymes they encode, i.e. glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase (SOD), heat shock protein (HSP70) and cytochrome P450 (CYP1A).

Common carp eggs
The biological material utilized in this study comprised C. carpio hatchlings obtained following egg incubation.Prior to exposure to nanoparticle solutions, the C. carpio eggs underwent incubation without prior contact with nanoparticles or toxins.The preparation of the biological material followed the protocol outlined by Sielska et al. (2022).Fertilization was conducted in Wojnarowicz solution, a standard medium for fish egg preparation.After fertilization (i.e., mixing the eggs with sperm and rinsing), the eggs were segregated into groups of 50 in nanoparticle-free water.Subsequently, the eggs underwent approximately 3 hours of soaking in water to facilitate swelling before the introduction of NPs (colloidal Cu, Cu-NPs and CuO-NPs).The biological material was incubated in NP suspensions during the swelling phase, a period when eggs are most susceptible to environmental toxins.After swelling, the eggs were rinsed and distributed into baskets within a closed circuit water system and left to hatch.Following hatching, C. carpio larvae were preserved in alcohol (Sielska et al. 2022).The control group did not undergo treatment with Cu-NPs, colloidal Cu or CuO-NPs.Ethical approval was not required to conduct the study.
Working concentration of 1 µg L −1 was prepared.The suspension was sonicated in an ultrasonic bath for 30 min, and vortexed before each use (Kowalska-Góralska et al. 2019).

Real-time PCR
RNA was isolated from previously prepared biological material (C.carpio hatchlings) (13 samples in total: 2 controls, 4 CuNP, 3 Cu, 4 CuO) using the EXTRACTME Total RNA Kit (Blirt) according to the protocol provided by the manufacturer.Genomic DNA was removed using DNaseI plus Reaction Buffer, included in the EXTRACTME TotalRNA isolation kit (Blirt).Subsequently, reverse transcription was performed using the RevertAid First Strand cDNA Synthesis Kit (Thermofisher Scientific) according to the protocol provided by the manufacturer.The concentration and quality of RNA and cDNA was determined using a NanoDrop spectrophotometer (Thermofisher Scientific).
Each 10-µl real-time PCR reaction mixture contained: 6.6 µl of distilled water, 0.2 µl of forward and reverse primer, 2.0 µl of HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (SolisBiodyne) (volume added according to manufacturer's recommendations) and 1 µl of cDNA.The negative control contained 7.6 µl of distilled water, 0.2 µl of forward and reverse primers (synthesized by Genomed), 2.0 µl of HOT FIREPol EvaGreen qPCR Mix Plus (ROX) (SolisBiodyne), without cDNA addition.The following gene primers were used for the reaction: gpx, cyp1a and hsp70 (Scown et al. 2010), sod and cat (Fazelan et al. 2020) (Table I).The β-actin (Fazelan et al. 2020) and alpha elongation factor (ef1a) (Zhang et al. 2016) genes were used as reference genes (Table I).Real-time PCR was performed in a thermocycler (Biorad) with the temperature profile recommended by the manufacturer.The annealing temperature was set individually for each gene.
The relative expression of each analyzed gene normalized to the reference gene (2−∆Ct) was calculated for each treatment.In addition, Livak's comparative method (Livak & Schmittgen 2001) was used to calculate the fold change in gene expression normalized to reference gene, relative to control results.
Heat shock protein (HSP70) was assessed using a commercially available Heat Shock Protein Assay Kit (Fluorometric) (BioVision, K2024-100).The test procedure was conducted according to the manufacturer's recommendations of the HSP70 activity kit.

SOD and CAT assays
Spectrophotometric analysis.SOD activity was estimated using the method of Giannopolitis and Ries (1977) by inhibiting the photoreduction of nitro blue tetrazolium chloride (Sigma-Aldrich, 298-83-9).The reaction solution contained: 0.1 M KH 2 PO 4 /K 2 HPO 4 (pH 7.8), 1.3 µM riboflavin (Sigma-Aldrich, 83-88-5), 13 mM methionine (Sigma-Aldrich, 63-68-3), 63 µM nitro blue tetrazolium chloride, 0.1 mM EDTA and the supernatant.The reaction was initiated by light (50 µmol m −2 s −1 ) and the absorbance was measured at 560 nm after 15 min of irradiance.One unit of SOD activity was defined as the amount of the enzyme required to inhibit 50% reduction of NBT in comparison with the positive control.The activity was expressed as U mg −1 protein.
The CAT activity was checked using the method of Rao et al. (1996).The catalase activity was spectrophotometrically determined at 240 nm in mixture solution: 50 mM KH 2 PO 4 /K 2 HPO 4 buffer (pH 7.0), and 14.3 mM H 2 O 2 and enzyme extract.The purified CAT (Sigma-Aldrich, 9001-05-2) was used as a calibration standard.The CAT activity was expressed as U mg −1 protein.

Native-PAGE and activity staining of SOD and CAT.
Sample were run electrophoretically in native-PAGE gel (at 4°C and 150 V) (Laemmli 1970).

Protein assay
The protein content in the enzymatic extracts was assayed by Bradford's method (Bradford 1976), using bovine serum albumin (BSA) as a standard (Bradford 1976).

Superoxide anion and hydrogen peroxide content
All ROS assays (O 2 •− and H 2 O 2 ) were analyzed in the same crude homogenate.The samples were crushed to a fine powder in liquid nitrogen using laboratory mill ball and homogenized for 10 min in cold 50 mM Tris-HCl pH 7.5 with 2% (w/v) PVP (Sigma-Aldrich, 9003-39-8).The samples were centrifuged for 20 min at 14,000 × g at 4°C, and the resulting supernatants were taken for spectrophotometric analysis.
Extracellular O 2 •− level was estimated using the method described by Misra and Fridovich (1972).The reaction mixture consisted of 17 mM Tris HCl pH 7.5, 20 mM epinephrine (Sigma-Aldrich, 51-43-4) (in 0.5 M HCl) and the supernatant.The oxidation of epinephrine to adrenochrome was measured in the reaction solution at 480 nm for 1 min.The epinephrine extinction coefficient was ε = 4.02 Effects of Cu-NPs on oxidative stress in C. carpio mM −1 cm −1 .The results were expressed as relative units corresponds to the epinephrine oxidation rate in the extracts and calculated as nmol min −1 mg −1 FW).
Extracellular H 2 O 2 level was measured followed by the protocol of Velikova et al. (2000), and was measured in the reaction solution (2.5 mM KH 2 PO 4 /K 2 HPO 4 buffer pH 7.0, 0.5 M KI (Sigma-Aldrich, 7681-11-0) in 10 mM KH 2 PO 4 /K 2 HPO 4 buffer, pH 7.0, and the extract) at 390 nm.A standard curve was prepared using the hydroxy peroxide standard.The results were expressed as nmol H 2 O 2 mg −1 FW.

Statistical analysis
All of the biochemical and gene expression analysis were carried out in 13 samples containing 5 larvae: 2 samples -controls, 4 samples -CuNP, 3 samples -Cu, 4 samples -CuO in three technical replicates, and the final means are presented with their standard deviation (SD).One-way analysis of variance, ANOVA (Statistica for Windows v. 13.0, Stat-Soft Inc., Tulsa, OK, USA) were used for data analysis of different parameter.Data were checked for normality and homogeneity of variance, and met these criteria.Duncan's multiple range test or post hoc Tukey's (HSD) test were used for analysis of variance.

Gene expression
Cyp1a2 gene expression increased in samples treated with Cu-NPs, Cu and CuO.The highest expression was found in samples isolated from individuals exposed to Cu (Figure 1(b)).The hsp70 gene showed increased expression in samples from hatchlings treated with Cu, Cu-NPs and CuO-NPs (Figure 1(d)).The expression of sod and cat, however, decreased in all samples compared to controls (Figure 1(f,h)).For samples isolated from hatchlings incubated with CuO-NPs, the expression was similar to control (Figure 1(h)).
The expression of the gpx gene was found to be lower than in control for hatchlings incubated with Cu and Cu-NPs.The CuO-NPs-treated samples showed no changes compared to control (Figure 1 (j)).In the case of CuO-NPs, the expression of sod and cat, although lower than in control, was characterized by the highest increase compared to Cu and CuO-NPs.

Protein level and activity
Protein level of cyp1a and hsp70 are presented in Figure 2. The level of hsp70 was higher in Cu-NPs and CuO-NPs-treated samples, while it was comparable to control in Cu-treated samples.The level of cyp1a however, was lower than in control in all treatments.
The enzymatic activity of GPX, CYP1A, HSP70, SOD and CAT enzymes is illustrated by bar graphs (Figures 1 and 3).An increase in enzymatic activity was observed only for HSP70 in samples exposed to Cu-NPs and CuO-NPs.For Cu-treated samples, HSP70 activity did not change compared to control (Figure 1(c)).The activity of CYP1A, SOD, Cu/Zn SOD, CAT, CAT isoenzymes and GPX enzymes decreased in samples treated with Cu, Cu-NPs and CuO-NPs (Figures 1(a,e,g,i) and 3(a,b)).CAT-2 isoenzyme activity, despite being lower compared to control, was higher than that of CAT-1 (Figure 3(b)).

Free radical content
The level of free radicals for individual samples is illustrated by bar graphs (Figure 4).In all samples (Cu, Cu-NPs, CuO-NPs), an increase in both O 2 •− and H 2 O 2 was observed.O 2 •− levels for all samples remained consistently high compared to control (Figure 4(a,b)).For H 2 O 2 , the highest increase was observed for samples incubated in CuO-NPs, and the lowest for samples treated with Cu (Figure 4(b)).

Discussion
Copper, as one of the essential trace elements, plays a vital role in the growth and physiology of organisms, including fish, and its addition has been shown to stimulate growth and enhance hematological parameters and antioxidant capacity in species such as rainbow trout (Oncorhynchus mykiss) or snow trout (Schizothorax plagiostomus) (Afshari et al. 2021;Delavari et al. 2022).It is worth noting, however, that precise dosage control is essential.As highlighted by Dawood et al. (2020), the recommended dietary dosage of Cu-NPs for C. carpio ranges between 2.19 and 2.91 mg/kg (Dawood et al. 2020).Copper nanoparticles are increasingly prevalent in various sectors of everyday life and industry.A notable application, owing to their biocidal properties, is their incorporation into disinfectants, which may feature Cu-NPs-coated fibers serving as potential agents against waterborne pathogens (Sheikh et al. 2011;Ghuglot et al. 2021).Additionally, Cu-

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A. Sielska et al.Effects of Cu-NPs on oxidative stress in C. carpio NPs can be used as probes for detecting mercury ions, catalysts or nanowires in electronics (Mirsafaei et al. 2015;Zhou et al. 2019;Pang et al. 2021).However, it has been found that under specific external conditions (water reservoirs) or internal environment (low pH of gastric acid) copper nanoparticles release ions that are highly toxic to organisms, including aquatic ones, especially when ingested (Chen et al. 2006;Conway et al. 2015;Asif et al. 2021).The documented toxic effects of nanoparticles, including copper, are primarily attributed to the induction of oxidative stress (Martínez et al. 2021), leading to adverse alterations in the body, such as brain lesions, H+ ATPase activity, organ damages,

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A. Sielska et al. particularly liver, and increased mortality in fish species such as O. mykiss, C. carpio or D. rerio (Al-Bairuty et al. 2013;Song et al. 2015;Zhang et al. 2022).Despite limited data concerning C. carpio or its larvae, two earlier studies have demonstrated negative effects of Cu-NPs on adult organisms (Johari et al. 2020;Naeemi et al. 2020).Copper nanoparticles, when exceeding the optimal concentration in feed, induce toxic effects, such as increased mortality, weight loss, dose-dependent accumulation or alterations in biochemical parameters (Johari et al. 2020) et al. 2021).In the present study, an increase in hsp70 expression occurred in hatchlings exposed to Cu, Cu-NPs and CuO-NPs.While elevated expression of sod and cat was reported in other species like O. niloticus or D. rerio (Kaur et al. 2019;Abdel-Latif et al. 2021), the current investigation revealed a decrease in the expression of these genes in hatchlings exposed to Cu, Cu-NPs and CuO-NPs.In D. rerio, a decline in sod expression was noted with increasing doses of CuO-NPs, mirroring a similar dose-dependent alteration observed in Tilapia zillii and O. niloticus exposed to zinc nanoparticles (Zn-NP) (Saddick et al. 2017).Regarding the gpx gene, an increase in its expression was previously observed in Danio rerio larvae incubated with CuO-NPs at a dose of 1 µg/ml, while at higher concentrations, a reduced expression was recorded (Kaur et al. 2019).In the current study, a decrease in gpx gene expression was recorded in C. carpio hatchlings exposed to 1 mg dm −3 Cu-NPs and Cu, while those treated with CuO-NPs showed the same level of expression as the control hatchlings.Changes have also been previously observed in the expression of gpx, sod and cat genes in various organs of Gasterosteus aculeatus fed Tubifex tubifex exposed to CuCl 2 and CuO-NPs.In the intestines, the expression of sod and cat was lower than in controls, while gpx levels remained unchanged.Conversely, in the liver, there was an increase in the expression of cat and gpx, with no changes observed for sod (Lammel et al. 2020).Zhang et al. (2022) reported a decrease in SOD an GPX activities, and an increase in CAT activity in C. carpio liver; however, in the present study the activity levels of these enzymes were decreased.

Conclusion
In conclusion, gene expression levels in samples incubated with Cu and CuO-NPs were significantly higher than those incubated with Cu-NPs.Both cyp1a and hsp70 genes exhibited elevated expression compared to controls in samples incubated with Cu, CuO-NPs, and Cu-NPs.Conversely, the expression of sod, cat and gpx genes either decreased or remained unchanged compared to the control.Notably, hsp70 showed the highest expression among the genes studied.These findings suggest that Cu and CuO-NPs exert higher toxicity than Cu-NPs in C. carpio hatchlings.In terms of enzymatic activity, CYP1A, SOD, Cu/Zn SOD, CAT, CAT-1, CAT-2, and GPX enzymes exhibited lower activity in samples incubated with Cu, Cu-NPs, and CuO-NPs compared to the controls.However, HSP70 activity increased in hatchlings exposed to Cu-NPs and CuO-NPs.The presence of Cu, Cu-NPs, and CuO-NPs also caused the generation and increase of O 2 •− and H 2 O 2 , confirming the induction of oxidative stress by copper nanoparticles.These findings imply that Cu, CuNPs and CuONPs exposure induces oxidative stress in C. carpio hatchlings.Meanwhile, the expression of antioxidant genes indicated normal protective responses in fish to counteract and mitigate the effects of oxidative damage exerted by Cu, CuNPs and CuONPs in their tissues.
Overall, copper nanoparticles induce oxidative stress by generating ROS, which can adversely impact fish organisms at the molecular and cellular Effects of Cu-NPs on oxidative stress in C. carpio levels.Nanoparticle-induced ROS generation via Fenton-type reactions prompts the mobilization of antioxidant and stress mechanisms in the body (Manke et al. 2013), as evidenced by the increased expression and activity of cyp1a and hsp70 observed in the present study.At high concentrations, nanoparticles, such as copper, can disrupt the delicate balance between ROS and antioxidants.This disturbance can potentially overload the protective capacity of the antioxidant mechanisms, resulting in lower expression and activity of enzymes involved in this process.A compromised antioxidant protection can consequently lead to further genotoxicity, cytotoxicity and other damage in fish such as C. carpio.Therefore, dose-and tissuedependent toxicity requires further investigations.The presented results lay the groundwork for a more comprehensive understanding of how copper nanoparticles impact various molecular parameters, as well as whole cells and tissues.

Figure 1 .
Figure 1.Effects of Cu, Cu-NPs or CuO (concentration 1 µg L −1 ) on enzymatic activity and transcribed level of cyp1a (a, b), hsp70 (c, d), sod (e, f), cat (g, h) and gpx (i, j).Relative expression was normalized by bactin and ef1a as reference transcripts.Vertical bars indicate ± SD.Duncan's multiple range test was used to test for significance of differences (p ≤ 0.05) in enzymatic activity.Statistical analyses of gene expression were performed using a post hoc Tukey's (HSD) test with a confidence interval of 0.05 and a confidence level of 95%.Differences between the mean values were considered to be significant at p < 0.01 or p < 0.05.Mean values with different letters (a-d) are significantly different (p < 0.05).

Figure 2 .
Figure 2. Effects of Cu, Cu-NPs or CuO (concentration 1 µg L −1 ) on gene expression reveiled as protein level of CYP1A (a) and HSP70 (b).Vertical bars indicate ± SD.Duncan's multiple range test was used to test for significance of differences (p ≤ 0.05).Mean values with different letters (a-b) are significantly different (p < 0.05).

Figure 3 .
Figure 3.Effect of Cu, Cu-NPs or CuO-NPs (concentration 1 µg L −1 ) on Cu/Zn-SOD (a) and CAT (b) isoenzyme activity.Vertical bars indicate ± SD.Duncan's multiple range test was used to test for significance of differences (p ≤ 0.05).Mean values with different letters (a-d) are significantly different (p < 0.05).