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. 2026 May 5;46(10):3632–3641. doi: 10.1002/jat.70231

Xenobiotic Metabolizing Enzyme Response of Rainbow Trout ( Oncorhynchus mykiss ) to Benzophenone‐3 and N,N‐diethyl‐m‐toluamide

Azra Bozcaarmutlu‐Büken 1,✉, Ahmet Günbür 1, Canan Sapmaz 1, Volkan Yıldırım 1, Hakan Türker 2
PMCID: PMC13532813  PMID: 42083724

ABSTRACT

Benzophenone‐3 (BP‐3) and N,N‐diethyl‐m‐toluamide (DEET) are ingredients of personal care products. Despite the frequent occurrence of both compounds in aquatic environments and the fact that fish are exposed to these chemicals together, their combined effects on xenobiotic metabolizing enzymes and antioxidant enzyme activities have not been studied in aquatic organisms. The aim of this study was to determine the effects of the combination of BP‐3 and DEET on cytochrome P450 and antioxidant system in rainbow trout ( Oncorhynchus mykiss ). For this purpose, juvenile male rainbow trout samples were treated with two different doses (100 and 500 μg/L) of BP‐3, DEET, and their combinations for 9 days. Low dose BP‐3 + DEET administration increased EROD activity. High‐dose BP‐3 and high dose BP‐3 + DEET administration reduced EROD and PROD activities. Glutathione S‐transferase activities of treatment groups were not affected by either individual exposure or co‐administration of BP‐3 and DEET. Individual administration low dose DEET increased catalase activity, whereas high dose BP‐3 increased glutathione reductase activity. The results indicated that cytochrome P450 enzyme activities were affected by the combined administration of BP‐3 and DEET. These enzymes are responsible for the metabolism of endogenous molecules and environmental pollutants. Accordingly, exposure to BP‐3 and DEET mixture may affect the metabolism of endogenous molecules and other pollutants in fish. Alteration of these enzyme activities highlights the toxicity of BP‐3 and DEET for fish and the need to improve strategies for monitoring and removing these contaminants from aquatic environments.

Keywords: antioxidant; benzophenone‐3 (BP‐3); cytochrome P450 (CYP); N,N‐diethyl‐m‐toluamide (DEET); rainbow trout ( Oncorhynchus mykiss ); xenobiotic metabolizing enzymes

Short abstract

Benzophenone‐3 (BP‐3) and N,N‐diethyl‐m‐toluamide (DEET) are ingredients of personal care products. In this study, rainbow trout samples were treated with two different doses (100 and 500 μg/L) of BP‐3, DEET, and their combinations for 9 days. The results indicate that BP‐3 and DEET individually and/or in combination modified certain xenobiotic metabolizing enzymes and antioxidant enzyme activities. Accordingly, the metabolism of endogenous molecules and other pollutants may be affected in rainbow trout.

1. Introduction

Sunscreens and insect repellents are two important cosmetic products whose use is increasing worldwide, especially in summer. Although these products contain many ingredients, two of them are quite remarkable. These are benzophenone‐3 (2‐hydroxy‐4‐methoxybenzophenone, BP‐3) and N,N‐diethyl‐m‐toluamide (N,N‐diethyl‐3‐methylbenzamide, DEET). BP‐3 is one of the UV filters found in sunscreen agents and cosmetics (Grabicova et al. 2013). It protects humans from the adverse effects of sunlight. It is a photostable and lipophilic chemical (Kim and Choi 2014). DEET is the main ingredient of insect repellents. It exerts its effect on insects by inhibiting acetylcholinesterase activity (Corbel et al. 2009).

Due to the extensive usage of insect repellents containing DEET and sunscreens containing BP‐3, both chemicals have been frequently detected in aquatic systems (Kim and Choi 2014; Downs et al. 2016; Dsikowitzkya et al. 2018; Mao et al. 2019; Cadena‐Aizaga et al. 2020; Painting et al. 2021; Wang et al. 2021; Cuccaro et al. 2022; Scheele et al. 2023; Bialkowska‐Jelinska et al. 2025; Motuźová et al. 2025). Recent studies have shown that DEET and BP‐3 can reach to mg/L levels in some aquatic environments (Painting et al. 2021; Scheele et al. 2023; Bialkowska‐Jelinska et al. 2025; Motuźová et al. 2025). It is clear that non‐target organisms are at the risk of co‐exposure to both of these chemicals together in aquatic systems.

It has been reported that BP‐3 alone can lead to genotoxic and mutagenic effects in fish (Almeida et al. 2019). It causes impaired gametogenesis and an increase in plasma vitellogenin in male fish (Coronado et al. 2008; Kinnberg et al. 2015). In addition, BP‐3 disturbs steroidogenic and hormonal pathways in adult male zebrafish ( Danio rerio ) (Blüthgen et al. 2012). Upregulation of the expression of cytochrome P450 gene (CYP1A) and glutathione S‐transferase (GST) has been reported by transcriptome analyses of zebrafish ( D. rerio ) larvae exposed to BP‐3 (Meng et al. 2020). Upregulation of cytochromes P450 enzymes (CYP2J and CYP4B1) and GST has been reported by transcriptome analyses of gilthead sea bream ( Sparus aurata ) exposed to DEET (Colás‐Ruiz et al. 2023).

Cytochrome P450 enzymes (CYPs) are responsible for the metabolism of both molecules. Benzophenone‐3 is mainly metabolized by CYP1A, CYP2B, CYP2C, and CYP2D in humans and CYP1A, CYP2C, and CYP3A in rats (Okereke et al. 1994; Watanabe et al. 2015). DEET is metabolized into two products: N,N‐diethyl‐m‐hydroxymethylbenzamide (BALC) and N‐ethyl‐m‐toluamide (ET) (Sudakin and Osimitz 2010). The BALC metabolite of DEET is produced by the oxidation reactions of CYP2B6 and CYP1A2 in humans (Usmani et al. 2002). The ET metabolite of DEET is produced by the N‐dealkylation reactions of CYP2C19, CYP3A4, CYP3A5, and CYP2A6 (Usmani et al. 2002). CYPs catalyze the metabolic reactions of endogenous (e.g., fatty acids, retinols, cholesterol, and testosterone) and exogenous (e.g., drugs, alcohols, solvents, polyaromatic hydrocarbons, polychlorinated biphenyls, and dioxins) molecules (Nebert and Gonzalez 1987; Guengerich 1990; Sen and Arinc 1998; Nelson 2003; Arinç and Bozcaarmutlu 2003; Bozcaarmutlu et al. 2014; Sapmaz et al. 2020; Esteves et al. 2021). These reactions can convert molecules into their less toxic forms (detoxification reactions) or more toxic forms (bioactivation reactions). Exposure to BP‐3 and DEET together may produce additive, synergistic or antagonistic effects on the CYP activities in fish; accordingly, metabolism of hormones or other endogenous molecules, and as well as pollutants and other exogenous molecules may altered. In addition, combined exposure to BP‐3 and DEET may alter important detoxification enzyme activities such as GST and antioxidant enzyme activities in fish. GSTs catalyze the conjugation reactions of reduced glutathione (GSH) and many xenobiotics (Tew and Ronai 1999). Reactive oxygen species (ROS) are formed during the metabolism of xenobiotics. Antioxidant enzymes including catalase, superoxide dismutase, glutathione reductase (GR), and glutathione peroxidase remove these reactive species from living organisms (Vilchis‐Landeros et al. 2024). Modulation of these activities by the combination of BP‐3 and DEET may cause the accumulation ROS and impair detoxification of other toxic pollutants in fish.

In recent years, UV filter and insect repellent chemicals have been combined into a single product for practical reasons. The existence of these combo products has increased the importance of examining the combined effects of these chemicals. In this study, our aim was to determine the combined effects of BP‐3 and DEET on cytochrome P450 related activities: 7‐ethoxyresorufin O‐deethylase activity (EROD) and 7‐penthoxyresorufin O‐depenthylase activity (PROD), GST and antioxidant enzyme activities such as catalase (CAT), and GR in the liver of rainbow trout ( Oncorhynchus mykiss ). Rainbow trout was chosen as the test organism because of its economic importance, widespread distribution, and proven suitability as model organisms in toxicology studies. In addition, it has been shown to be more sensitive to DEET than the other fish species (Office of Pesticide Programs 2000; Costanzo et al. 2007). Sexually immature male fish were used in this study to determine the changes in important xenobiotic metabolizing enzyme activities before the reproductive period.

2. Materials and Methods

2.1. Animal Treatments

Juvenile male rainbow trout ( O. mykiss ) samples were obtained from local fish farm in Bolu. The fish were kept in separate pond for 3 days and then transferred to the Fish Biology Laboratory of Bolu Abant Izzet Baysal University. They were randomly divided into eight groups as control, solvent control (DMSO), 100 μg/L BP‐3, 100 μg/L DEET, 100 μg/L BP‐3 + 100 μg/L DEET, 500 μg/L BP‐3, 500 μg/L DEET, and 500 μg/L BP‐3 + 500 μg/L DEET. The average fish weight and length for 96 rainbow trout were 26.96 ± 0.81 g (average weight ± SEM) and 13.76 ± 0.13 cm (average length ± SEM), respectively. BP‐3 and DEET were purchased from Sigma‐Aldrich (Saint Louis, Missouri, USA). The doses of the chemicals were selected according to previous studies conducted in fish and the high levels reported in aquatic systems, allowing for the assessment of potential maximum effects over a short period (Slaninova et al. 2014; Ortiz‐Román et al. 2024). Chemicals were dissolved in DMSO, the concentration of DMSO was 0.001% in all treatment groups.

Fish were kept at 10°C–12°C, pH 7.5–7.8, in pre‐filtered and aerated freshwater (> 85% saturation) in 200‐L circular fiberglass tanks under static conditions and a 12/12 h light–dark cycle throughout the experimental period. Fish were not fed during the experimental period to avoid deterioration of water quality. Furthermore, variations due to feeding were eliminated, as BP‐3 has been reported to alter feeding habits and weight gain in fish (Zhang et al. 2023). Tank water was exchanged by replacing three‐quarters of the volume every 12 h with repeating the chemical administration for 9 days. At the end of the treatment period, fish were anesthetized by immersion in an ice bath and then killed by decapitation. Liver tissues were taken and placed in freezer storage bags. Due to the small size of livers, they were combined. Samples were initially stored in liquid nitrogen and subsequently at −80°C. The Animal Experiments Local Ethics Committee of Bolu Abant Izzet Baysal University approved this study with process number of 2017/28.

2.2. Preparation of Microsomal and Cytosolic Fractions of Rainbow Trout Liver

Rainbow trout liver microsomes and cytosols were prepared by differential centrifugation as described by Arinç and Şen (1993), with minor modifications. All solutions, tissues, and tissue fractions were kept in an ice bath throughout the procedure. Liver tissues were homogenized in homogenization buffer containing 1.15% KCl, 1.0 mM EDTA (pH 7.4), 0.25 mM ε‐ACA, and 0.1 mM PMSF. The tissues were homogenized in this solution at a volume of 7.5 times the tissue weight. The homogenized tissue was first centrifuged at 10,000 ×g for 20 min. The resulting supernatant was centrifuged at 105,000 ×g for 1 h using an ultracentrifuge. The supernatant (cytosolic fraction) was taken into glass flasks, divided into small aliquots, and placed into Eppendorf tubes. Aliquots of cytosols were stored at −80°C. The pellet was washed with a solution containing 1.15% KCl and 1.0 mM EDTA (pH 7.4) and centrifuged at 105,000 ×g for 50 min. The final pellet (microsomal fraction) was resuspended in a 10% glycerol solution containing 1.0 mM EDTA (pH 7.4). Microsomes were separated into small aliquots and placed into Eppendorf tubes. Aliquots of microsomes were stored at −80°C. Protein concentrations of the microsomal and cytosolic fractions were determined using Lowry method with bovine serum albumin as the standard (Lowry et al. 1951).

2.3. Enzyme Activity Measurements

CYP1A‐associated 7‐ethoxyresorufin O‐deethylase (EROD) and CYP2B‐associated 7‐pentoxyresorufin O‐depenthylase (PROD) activities were measured in rainbow trout liver microsomes according to the method described by Burke and Mayer (1974), with slight modifications. A typical reaction mixture contained 100 mM potassium phosphate buffer (pH 7.8), 100 mM NaCl, 1.2 mg/mL BSA, 0.5 mM NADPH generating system, 200 μg fish liver microsome, and 6.5 μM substrate (7‐ethoxyresorufin for EROD and 7‐pentoxyresorufin for PROD) in a 2.0 mL final volume. The formation of 7‐hydroxyresorufin was measured for 5 min for EROD and 10 min for PROD by using a spectrofluorometer (Hitachi F‐4500, Hitachi High‐Technologies Corporation, Tokyo, Japan) at 535 nm (excitation) and 585 nm (emission) wavelengths. Resorufin was used as a standard. Enzyme activity was expressed as the formation of 7‐hydroxyresorufin per milligram of protein per minute.

GST activities were measured as described by Habig et al. (1974), using a reaction mixture containing 100 mM potassium phosphate buffer (pH 7.4), 3.0 mM GSH, 0.15 mL enzyme source (25‐ to 100‐fold diluted cytosols) and 1.0 mM CDNB in 2.0 mL reaction mixture. The formation of 1‐glutathione‐2,4‐dinitrobenzene was followed at 340 nm for 2 min using a Hitachi U‐2900 spectrophotometer. GST activity was calculated using 9.6 mM−1 cm−1 as an extinction coefficient (ε 340).

Catalase activities were determined according to the method of Aebi (1984). Fish liver cytosols were pretreated with 1% Triton X‐100 (1:10) for 10 min. A typical reaction mixture contained diluted cytosol, 50 mM potassium phosphate buffer (pH 7.0), and 50 mM H2O2 in a final volume of 3.0 mL. The reaction was measured at 240 nm for 1 min using a Hitachi U‐2900 spectrophotometer. Enzyme activities were calculated using 0.0364 mM−1 cm−1 as an extinction coefficient.

GR activities were measured in fish liver cytosols according to the procedure of Carlberg and Mannervik (1985). A typical reaction mixture contained 100 mM of potassium phosphate buffer (pH 7.0), 0.5 mM of EDTA (pH 7.0), 0.1 mM of NADPH, fish liver cytosol, 1.0 mM oxidized glutathione (GSSG), and distilled water in a final volume of 2.0 mL. The reaction was measured at 340 nm for 5 min using a Hitachi U‐2900 spectrophotometer. Enzyme activities were calculated using 6.22 mM−1 cm−1 as an extinction coefficient (ε 340).

2.4. Statistical Analysis

Enzyme activities were expressed as the average of enzyme activities ± standard error of mean (SEM). The Shapiro–Wilk test was used to assess the normality of the data. EROD activities were log‐transformed to achieve a normal distribution. Then EROD, PROD, CAT, and GR enzyme activities were analyzed using the LSD post hoc test. GST (non‐parametric) data were first analyzed using the Kruskal–Wallis test, and then Dunn's test was employed. p‐value equal to and less than 0.05 was considered statistically significant unless otherwise specified. The statistical analysis was performed using the SPSS statistical package (SPSS Statistics 21.0, Chicago, IL).

3. Results

In this study, the effects of BP‐3 and DEET were investigated using eight experimental groups, including a control group, a solvent (DMSO) control group, 100 and 500 μg/L BP‐3, DEET, and their combination groups. In this study, none of the fish died as a result of the chemical treatments. Sexually immature male fish were used to minimize hormonal fluctuations associated with reproduction. Fish whose gallbladders were ruptured during liver removal were excluded from the groups. The total number of fish used in the analysis was 96. Fish livers were combined due to the small size of the fish. The number of individuals in each group and the number of pools obtained by combining livers were given in Table 1. Enzyme activities were measured in duplicates or sometimes in triplicates.

TABLE 1.

Biomarker enzyme activities in the liver of rainbow trout ( Oncorhynchus mykiss ).

N (n) EROD (pmol/min/mg protein) PROD (pmol/min/mg protein) GST (nmol/min/mg protein) CAT (μmol/min/mg protein) GR (nmol/min/mg protein)
Control 14 (5) 12.24 ± 1.08 1.67 ± 0.12 1052 ± 131 46 ± 4 29.39 ± 3.33
DMSO (solvent control) 12 (4) 11.18 ± 0.35 1.92 ± 0.10 944 ± 57 117 ± 13 36.60 ± 5.08
100 μg/L BP‐3 12 (4) 7.63 ± 0.43 1.89 ± 0.08 950 ± 115 180 ± 48 27.22 ± 2.85
100 μg/L DEET 14 (5) 9.63 ± 0.41 1.98 ± 0.23 919 ± 78 310 ± 31 32.85 ± 1.12
100 μg/L BP‐3 ± 100 μg/L DEET 11 (4) 25.99 ± 8.58 2.08 ± 0.26 773 ± 54 81 ± 4 32.78 ± 2.85
500 μg/L BP‐3 7 (4) 5.49 ± 0.46 1.08 ± 0.05 802 ± 105 49 ± 5 54.60 ± 9.45
500 μg/L DEET 13 (4) 12.88 ± 1.39 1.96 ± 0.29 968 ± 75 109 ± 29 37.80 ± 6.61
500 μg/L BP‐3 ± 500 μg/L DEET 13 (6) 5.27 ± 0.35 1.18 ± 0.18 863 ± 37 102 ± 12 36.87 ± 4.43
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Note: N = number of fish in the group, n = number of pool prepared from the fish livers present in the group. The pools were prepared by combining 2–3 fish livers except 500 BP‐3 group in which only one fish liver sample containing pools were present.

EROD and PROD activities were measured in microsomal fractions. EROD activities of the control and DMSO groups were not significantly different from each other. Highly elevated EROD activity was measured in the group treated with 100 μg/L BP‐3 + 100 μg/L DEET (25.99 ± 8.58 pmol/min/mg protein). The EROD activities measured in this group were significantly higher than those of the control, the solvent control (DMSO), and the other groups (Figure 1A). The lowest EROD activity was measured in the 500 μg/L BP‐3 + 500 μg/L DEET co‐administration group (5.27 ± 0.35 pmol/min/mg protein). The EROD activities of this group were significantly lower than those of the control, the solvent control (DMSO), low dose BP‐3, low dose DEET, low dose BP‐3 + DEET, and high dose DEET groups.

FIGURE 1.

FIGURE 1

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Cytochrome P450 (CYP)‐associated enzyme activities in rainbow trout liver microsomes. (A) CYP1A‐associated 7‐ethoxyresorufin O‐deethylase and (B) CYP2B‐associated 7‐pentoxyresorufin O‐depenthylase activities of rainbow trout liver microsomes. Square brackets above the bars indicate significant differences between groups (p ≤ 0.05).

The highest PROD activity was measured in the 100 μg/L BP‐3 + 100 μg/L DEET group (2.08 ± 0.26 pmol/min/mg protein). However, this activity was not significantly different from the solvent control (DMSO) and the control groups (Figure 1B). The groups treated with 500 μg/L BP‐3 and 500 μg/L BP‐3 + 500 μg/L DEET exhibited significantly lower PROD activities compared with the control, the solvent control (DMSO), low dose BP‐3, low dose DEET, low dose BP‐3 + DEET, and high dose DEET.

GST, catalase, and GR activities were measured in cytosolic fractions. The lowest GST activities were determined in the 100 μg/L BP‐3 + 100 μg/L DEET group (773 ± 54 nmol/min/mg protein) (Table 1). However, this activity was not significantly different from the other groups. GST activity was not significantly affected by the administration of DEET, BP‐3, and their combinations.

Catalase activities of the solvent control (DMSO) group were significantly higher than those of the control (p = 0.035) (Figure 2A). Highly elevated catalase activities were measured in the 100 μg/L DEET administration group (310 ± 31 μmol/min/mg protein). These activities were significantly higher than the catalase activities of all the other groups (Figure 2A). Catalase activities of the 100 μg/L BP‐3 administration group were also high (180 ± 48 μmol/min/mg protein). These activities were significantly higher than those of the control, 100 μg/L BP‐3 + 100 μg/L DEET, 500 μg/L BP‐3, 500 μg/L DEET, and 500 μg/L BP‐3 + 500 μg/L DEET administration groups. However, this activity was not significantly different from those of the solvent control (DMSO) group (p = 0.071). The lowest catalase activities were measured in the 500 μg/L BP‐3 administration group (49 ± 5 μmol/min/mg protein). However, these activities were not significantly different from that of the solvent control (DMSO) group (p = 0.054). Catalase activities of the 500 μg/L DEET and 500 μg/L BP‐3 + 500 μg/L DEET administration groups were higher than that of the control group, but these differences were not statistically significant (p = 0.058 and p = 0.061, respectively).

FIGURE 2.

FIGURE 2

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Antioxidant enzyme activities in rainbow trout liver cytosols. (A) Catalase and (B) glutathione reductase activities. (*) Different from all groups. Square brackets above the bars indicate significant differences between groups (p ≤ 0.05).

Highly elevated GR activities were measured in the 500 μg/L BP‐3 administration group (54.60 ± 9.45 nmol/min/mg protein). GR activities in this group were significantly higher than in all the other groups (Figure 2B).

4. Discussion

BP‐3 and DEET are lipid‐soluble chemicals with low degradation rates in the aquatic environment. Their levels are high in some aquatic systems due to their intensive use and low degradation rates (Painting et al. 2021; Scheele et al. 2023; Bialkowska‐Jelinska et al. 2025; Motuźová et al. 2025). Fish are exposed to these chemicals together. However, the combined effects of BP‐3 and DEET on fish have not yet been reported. Exposure to BP‐3 and DEET together may cause synergistic, additive or antagonistic effects on enzyme activities in fish and their co‐occurrence in aquatic habitats may increase ecological risks. In this study, the combined effects of them on xenobiotic metabolizing and antioxidant enzyme activities were determined in sexually immature male fish. Previous studies have shown that BP‐3 affects the synthesis of certain hormones in fish (Blüthgen et al. 2012; Coronado et al. 2008; Kinnberg et al. 2015). Hormonal changes have also been reported in mature fish especially during the spawning season (Scott et al. 1984; Bennetau‐Pelissero et al. 2001; Estay et al. 2012). Differences in the synthesis of certain proteins, including cytochrome P450s, have been reported in fish during the spawning season (Lange et al. 1999; Thibaut and Porte 2004; Uno et al. 2012). In this study, differences related to the reproductive period were minimized by using juvenile male fish.

The role of cytochrome P450 system in the metabolism of both BP‐3 and DEET has been reported in humans and rats (Usmani et al. 2002; Nakagawa and Suzuki 2002; Watanabe et al. 2015; Mutlu et al. 2020). However, the metabolism of BP‐3 and DEET may differ in fish, as some cytochrome P450s are not expressed in fish species. CYP1A is commonly used as a biomarker, particularly in environmental monitoring studies (Goksøyr 1995; Arinç et al. 2000; Bucheli and Fent 1995; Bozcaarmutlu et al. 2015). It is responsible for the metabolism of both exogenous and endogenous molecules (Lu et al. 2020). CYP1A level is commonly determined by measuring EROD activity. In this study, significantly elevated EROD activities were measured in low dose BP‐3 + DEET co‐administration group. However, a reduction in the EROD activity was determined at high dose DEET + BP‐3 administration group compared with single exposures, the solvent control (DMSO), and the control groups. The effect of BP‐3 alone on CYP1A‐associated EROD activity has been studied in goldfish ( Carassius auratus ) (Zhang et al. 2020). In that study, EROD activity increased with 200 μg/L BP‐3 on the 7th day of exposure but decreased to the control level by the 28th day. In another study, EROD activity increased with 100 μg/L BP‐3 on the 14th day of exposure in zebrafish ( D. rerio ) (Sun et al. 2022). Transcriptome analyses of gilthead sea bream ( S. aurata ) have shown that the CYP1A‐related aryl hydrocarbon receptor nuclear translocator protein (ARNT) gene was upregulated following 10 μg/L DEET exposure for 22 days (Colás‐Ruiz et al. 2023). In the current study, individual administration of DEET did not alter EROD activities, whereas EROD activities were reduced by BP‐3. However, CYP1A‐associated EROD activity was synergistically increased in juvenile rainbow trout following exposure to low dose BP‐3 + DEET mixture. This result indicates that BP‐3 and DEET have a modulatory role in the regulation of CYP1A activity. In addition, the reduction in CYP1A‐associated activity in high dose BP‐3 + DEET administration group may result from competition between two substrates for CYP1A enzyme, enzyme saturation, or accumulation of metabolic products. Induction of CYP1A‐associated activity by low dose BP‐3 + DEET mixture may increase the metabolism of endogenous molecules; as a result, the synthesis and elimination of these molecules may also increase. Some of the pollutants are converted into more toxic forms by CYP1A. Thus, the generation of toxic metabolites may be triggered by increased CYP1A activity. Reduction of CYP1A activity by high dose of BP‐3 + DEET mixture may lead to the accumulation of pollutants in fish.

The other cytochrome P450‐related activity examined in this study was PROD activity which is associated with CYP2B (Franco et al. 2025). CYP2B is responsible for the metabolism of xenobiotics and endogenous compounds such as arachidonic acid, lauric acid, testosterone, and estradiol in humans (Mo et al. 2009). In the current study, PROD activity was not significantly altered at 100 μg/L BP‐3, 100 μg/L DEET, or their combinations. The effect of 100 μg/L BP‐3 on PROD activity was reported in zebrafish ( D. rerio ) (Sun et al. 2022). PROD activity increased at this dose in that study. In the current study, the lack of change in PROD activities at low doses may result from adaptation of fish to this dose of chemicals during the 9‐day experimental period. However, significantly lower PROD activities were measured in the high dose BP‐3 and BP‐3 + DEET co‐administration groups in juvenile male rainbow trout. This indicates that CYP2B‐associated PROD activity was inhibited in the high doses of BP‐3 and BP‐3 + DEET. CYP2B is generally found in detoxification reactions in humans (Mo et al. 2009). Reduction in this activity as a result of exposure to BP‐3 and combination of BP‐3 and DEET may reduce the detoxification reactions of toxic chemicals and synthesis of hormones in fish.

GST is one of the phase II enzymes involved in the detoxification reactions of many xenobiotics. In this study, GST activity was not significantly affected by individual or combined exposure to DEET and BP‐3. GST and antioxidant enzyme activities have also been measured in the liver of common carp ( Cyprinus carpio L.) exposed to 1 μg/L DEET, 0.1 mg/L DEET, and 1 mg/L DEET for 28 days (Slaninova et al. 2014). Similarly, no significant changes were reported in GST activity or other antioxidant enzyme activities in that study. However, transcriptome analyses have indicated that the gene coding GST was upregulated in gilthead sea bream ( S. aurata ) exposed to 10 μg/L DEET. Induction of GST has also been shown by BP‐3 in fish. In a study, significantly high GST activities were measured in Zebrafish ( D. rerio ) treated with 100 μg/L BP‐3 (Sun et al. 2022). In another study, the effect of a BP‐3 containing mixture was measured in tilapia ( Oreochromis niloticus ) (Sovierzoski et al. 2023). In that study, GST activity was not significantly affected by BP‐3 exposure. Similarly, the results of the current study showed that individual administrations of DEET and BP‐3, as well as their co‐administration, did not alter GST activity, even at high concentrations, in rainbow trout. This result may arise from adaptation of GST activity in rainbow trout to these chemicals at these doses.

Antioxidant enzyme activities were also measured in this study. Catalase activities of the DMSO group were significantly higher than that of the control. DMSO is widely used as a solvent in toxicology studies. However, the results of this study suggest that DMSO exposure may induce oxidative stress in juvenile fish. The effect of BP‐3 on catalase activity has been reported in the liver of goldfish ( C. auratus ) (Zhang et al. 2020). Catalase activity increased at 200 μg/L on the 7th day of exposure but returned to its control levels on the 28th day of exposure. In another study, DEET did not significantly alter catalase activity in common carp ( C. carpio L.) after 28 days of exposure (Slaninova et al. 2014). In the current study, low dose BP‐3 and DEET administration alone increased catalase activity, indicating increased ROS production in juvenile male fish. However, catalase activities in the low dose BP‐3 + DEET co‐administration group were significantly lower than in the low dose individual administration groups. This decrease may result from excessive ROS production and inhibition of catalase enzyme activities with ROS.

Another antioxidant enzyme activity measured in this study was GR. GR activities in low dose and high dose BP‐3 + DEET co‐administration groups were not significantly different from that of the solvent control and control groups. However, GR activity was significantly higher in the high dose BP‐3 administration group compared with all the other groups. GR activity is responsible for maintaining glutathione homeostasis and defending against oxidative stress (Vašková et al. 2023). An increase in this activity may indicate a disruption in homeostasis and the antioxidant system. GR activity measurements have been reported in common carp ( C. carpio L.) treated with DEET. In that study, GR activities were not affected by DEET treatment in common carp (Slaninova et al. 2014). Similarly, GR activities were not affected by individual administration of DEET in the current study.

The results of this study showed that cytochrome P450s involved in detoxification and activation of xenobiotics are affected by exposure to BP‐3 and DEET mixture in juvenile male fish before the reproductive period. There were differences in certain enzyme activity results in individual administration of BP‐3 and DEET between the current study and the studies carried out in different species. This may arise from the sensitivity difference between species to contaminants. The other factor may be related to the developmental stage of the fish used. The expression of the enzymes differs at different developmental stages.

5. Conclusions

In this study, for the first time, the combined effects of BP‐3 and DEET, two widely used ingredients in personal care products, were investigated on xenobiotic metabolizing and antioxidant enzyme systems in fish. These two chemicals are frequently detected in aquatic systems, and fish are exposed to them. The results of this study indicated that the combined administration of these chemicals modified certain CYP activities. These enzymes are responsible for the metabolism of endogenous and exogenous molecules. Accordingly, the metabolism of both endogenous and exogenous molecules may be affected in fish exposed to a BP‐3 and DEET mixture. As a result of this, the hormonal activities of fish may be disrupted, and/or other toxic pollutants may accumulate in fish. The results of this study highlight their potential ecological risks. Further studies are needed to clarify the effects of BP‐3 and DEET mixtures by investigating both sexes, longer exposure times, and fish at different developmental stages. Recently, UV filter chemicals and insect repellents have been used together by humans in combo preparations for practical reasons. It is clear that special attention should be given to the content of such preparations and their combined effects.

Funding

This research was supported by Bolu Abant Izzet Baysal University (Project No: BAP‐ 2019.03.03.1406).

Conflicts of Interest

The authors declare no conflicts of interest.

Acknowledgments

We would like to thank ChatGPT for its assistance in grammatical corrections in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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