The administration of methyl and butyl parabens interferes with the enzymatic antioxidant system and induces genotoxicity in rat testis: possible relation to male infertility

Abstract

Parabens are esters of p-hydroxybenzoic acid, used for decades as a preservative in many products, including agrochemicals, pharmaceuticals, foods and cosmetics. Concerns regarding parabens toxicity include adverse effects on endocrine activity, carcinogenesis, infertility, spermatogenesis, and adipogenesis. The present study aimed to investigate the in vivo administration of methyl and butylparaben at concentrations of 100 and 200 mg/kg body weight, by subcutaneous injection, in variable murinometric measurements, antioxidant systems and genotoxicity. The administration of parabens did not affect the consumption of water and food. However, there was a decrease in the weight of the testes and the seminal vesicle (p < 0.05). The administration of parabens caused an increase in superoxide dismutase for methylparaben (200 mg/kg) and both concentrations of butylparaben (p < 0.05). Catalase showed increased activity in all groups treated with parabens. In contrast, glutathione reductase and glutathione S-transferase suffered a decrease in the groups treated with both parabens. These results show that parabens, especially butyl, can affect the rat testis enzymatic antioxidant system, decreasing the cellular antioxidant capacity, which was confirmed by the decrease in the glutathione reducing power, expressed by the reduced glutathione/oxidized glutathione ratio. Therefore, an increase in lipid peroxidation was observed, which was significant in the case of butyl. Genetic Damage Indicator values show that butylparaben treatments displayed significantly higher values than the control. This study shows for the first time that parabens can induce genotoxicity in the rat male reproductive organ.

Keywords:

• Parabens
• antioxidant system
• DNA damage
• oxidative stress
• rats
• testis

1. Introduction

Parabens are a type of preservative widely used in the cosmetics, food, and pharmaceutical industries for more than 70 years (Fransway et al. 2019). Much has already been written about parabens’ toxicological effect, yet it remains a subject that is not consensual. The main argument for the use of parabens is that these compounds have been used and studied for a long time and that their replacement could result in their substitution with less proven and potentially unsafe alternatives (Petric et al. 2021). Although this is a valid argument, it should not stop further studies on the possible toxic effects of parabens and the development of compounds capable of replacing parabens.

Data from exposure to parabens based on real samples measurements in Chinese females show that humans can be exposed to 0.326 mg kg⁻¹ daily (Ma et al. 2016). Although parabens are suspected of contributing to infertility, so far a direct relationship between the concentration of parabens in urine and infertility in both women (Mínguez-Alarcón et al. 2016) and men (Adoamnei et al. 2018) has not yet been demonstrated. However, it is already clearly shown that exposure to parabens decreases spermatogenesis and motility of spermatozoa and will thus affect male fertility (Kang et al. 2002). Oxidative stress, induced by parabens, plays a central role in toxicity (Shah and Verma 2011), which directly translates into high levels of lipid peroxidation, probably as a consequence of the reduction in non-enzymatic and enzymatic antioxidants. These effects have already been observed in hepatocytes, under the action of butylparaben (Shah and Verma 2012) and in keratinocytes, when exposed to sunlight and the action of methylparaben (Handa et al. 2006). Exposure of female rats to parabens during pregnancy causes severe changes in mitochondrial bioenergetics and alterations in the antioxidant capacity of testicular germ cells of the F1 generation (Oliveira et al. 2020). In vitro studies show that long-chain parabens can induce susceptibility to the opening of the mitochondrial transient permeability pore in the presence of calcium (Martins et al. 2021). Parabens were detected in amniotic fluid samples, and there is a possible correlation between the exposure of pregnant women to parabens and the length at birth of newborns (Golestanzadeh et al. 2022).

Glutathione reduced (GSH), the main non-protein intracellular thiol compound, is known to protect against reactive radical species. Its depletion by parabens can be related to increased ROS production, which can induce DNA damage (Chung et al. 2005, Martín et al. 2010). Lymphocytes exposed to high concentrations of parabens caused DNA damage, analyzed by comet assay (mean tail length) (Bayülken et al. 2019, Todorovac et al. 2021). An overall positive correlation has been demonstrated between the mixture of chemicals used in personal care products and 8-hydroxy-2′-deoxyguanosine, with methylparaben and mono-benzyl phthalate contributing most to this association (Liao et al. 2022). Thus, due to the widespread existence of these chemicals in personal care products and the high frequency of use for humans, it is realistic to admit a synergistic or additive toxic effect.

2. Materials and methods

2.1. Chemicals

Methylparaben (CAS No. 99–76-3) and Butylparaben (CAS No. 94–26-8) were white powders with a purity of at least 99.5%, purchased from Sigma Chemical Co. (St. Louis, MO, USA). The solutions were prepared in absolute ethanol and diluted with peanut oil (Sigma-Aldrich, St. Louis, MO, USA) to obtain concentrations of 0 (control-oil), 100 and 200 mg/kg/d. These doses were selected based on data from the scientific literature, as well as the results of previous studies (Garcia et al. 2017, Oliveira et al. 2020).

2.2. Animals and experimental design

Young male Wister rats (6-week old) were obtained from Charles River (Barcelona, Spain), housed at 22 ± 2 °C under artificial light for 12-h light/day cycle and controlled humidity (40–60%) with access to water and food (Panlab rodent chow) ad libitum, and used throughout the experiments. Rats were randomly divided into five groups: control (CTRL), 100 mg/kg methylparaben (MP-100), 200 mg/kg methylparaben (MP-200), 100 mg/kg butylparaben (BP-100), and 200 mg/kg butylparaben (BP-200). For sub-chronic toxicity study, the paraben was subcutaneously administered in a volume of 0.5 mL, in the inguinal zone at doses of 100 and 200 mg/kg (n = 6/group/sex) daily to Wistar rats for 10 days. Animals were monitored daily by University Lab Animal Resources veterinary staff. The body weight of each rat was recorded daily to calculate the volume of each compound to be administered. Food and water consumption were also monitored daily. Three days after the last administration, body weights were recorded, and the animals were sacrificed using intraperitoneal ketamine and xylazine followed by cardiac puncture and exsanguination, as recommended by Federation of European Laboratory Animal Science Associations (FELASA). The experiments were carried out following the National (DL 129/92; DL 197/96; P 1131/97) and European Convention for the Protection of Animals used for Experimental and Other Scientific Purposes and related European Legislation (2010/63/EU).

2.3. Tissue preparation

After removal, testes were rinsed in 50 mM phosphate buffer, pH 7.4 and weighed. A 20% (w/v) testis homogenate was prepared with Ultra-Turrax homogenizer (IKA T25, Digital), followed by homogenization with a Potter-Elvehjem glass-Teflon homogeneizer. The crude homogenate was centrifuged at 800 xg for 10 min to precipitate nuclei and other cellular debris. The supernatant was centrifuged at 10 000 × g for 20 min to separate mitochondria. The supernatant obtained (postmitochondrial fraction) was used to access the antioxidant enzyme activity. The protein content of samples was estimated using bovine serum albumin as standard.

2.4. Determination of antioxidant enzymes

Superoxide dismutase (SOD) activity was evaluated using the xanthine-xanthine oxidase system. The reduction of nitroblue tetrazolium (NBT) was used as the detection molecule (560 nm). The reaction mixture contained potassium phosphate buffer (100 mM, pH 7.0), hypoxanthine (10 mM), and NBT (10 mM). The reaction was initiated by adding xanthine oxidase (0.023 U mol⁻¹) to enzymatic extract at 30 °C. Results are presented as U/min/mg of protein. Catalase (CAT) activity was evaluated by measuring hydrogen peroxide (H₂O₂) consumption at 240 nm. The reaction was carried out in a solution containing 100 mM phosphate buffer and 20 mM H₂O₂ at 30 °C. Results are expressed as μmol/min/mg protein. The activity of glutathione reductase (GR) was measured at 340 nm by reducing oxidized glutathione (GSSG, 1.0 mM) by NADPH (0.3 mM) in 50 mM phosphate buffer pH 7.0 containing 0.5 mM EDTA. The result was expressed as μM NADPH oxidized/min/mg of protein by using the extinction coefficient of NADPH (6220 M⁻¹ cm⁻¹). The activity of glutathione S-transferase (GST) was measured at 340 nm using reduced glutathione (GSH, 5 mM) and 1 chloro-2,4-dinitrobenzene (CDNB, 1 mM) as substrates. The result was calculated using the extinction value of 9.60 mM^–1 cm⁻¹ and was expressed as mM CDNB/min/mg of protein.

2.5. Quantification of oxidized and reduced glutathione content

Reduced glutathione (GSH) levels were spectrofluorimetrically measured in 100 mM phosphate buffer with 5 mM EDTA (pH 8.0), following derivatization with O-phthalaldehyde (OPA, 1 mg/mL) at 339 and 426 nm as excitation and emission wavelengths, respectively. After 30 minutes of incubation in 40 mM ethylmaleimide (NEM) and adjusting to pH 12 with 0.1 N of NaOH, oxidized glutathione (GSSG) levels were measured using the same procedure. A standard curve for GSH and GSSG was made. GSH and GSSG concentrations are presented as nmol/mg of protein, and Glutathione-reducing power was expressed as GSSG/GSH.

2.6. Lipid peroxidation

For quantifying lipid peroxidation, the method described by (Devasagayam et al. 2003) was used. This method is based on the oxidation of the ferrous ion (Fe²⁺) to ferric ion (Fe³⁺), in an acidic medium, using an orange xylenol dye that will be oxidized to a blue-purple complex with a maximum absorption between 550–600 nm.

The Fox reagent solution, prepared previously, consisting of xylenol orange dye (1 mM), H₂SO₄ (25 mM), di-tert-butylmethylphenol (BHT) ethanolic solution (4 mM), and ammoniacal ferrous sulfate (250 mM). The sample (1 mg/mL) was incubated for 30 minutes in a buffer (Hepes 5 mM, KCl 100 mM, pH = 7.12), at 30° C, in a final volume of 1 mL with occasional shaking. Then, 2 ml of methanol was added, with continuous stirring, until visualization of the membranes’ rupture. All solution was then centrifugated for 10 min at 1950 RCF (Sigma 2–16KL). Subsequently, 2 mL of the supernatant was removed, and 2.25 mL of Fox’s reagent was added, allowing reaction for 30 min for subsequent spectrophotometric reading at 570 nm (Varian Cary 50). The signal was read against the hydrogen peroxide standard curve, and the values obtained were expressed in µM H₂O₂/mg protein.

2.7. Genotoxicity assessment by the comet assay

Spermatozoa were obtained from the distal section of the epididymal tail, collected with a micropipette. The sperm fraction was diluted 1:20 in a tris-fructose-citric acid containing 15% glycerol. The alkaline comet assay was performed, according to Sipinen et al. (2010). The cells were counted, and a volume containing approximately 12 × 10⁴ sperm cells was removed and centrifuged, the supernatant discarded, and the pellet resuspended in 140 µL of cooled PBS and an equal volume of low melting point agarose (Merck KGaA, cat.n^o. A9414), at 2% w/v in PBS (final concentration 1%). Samples were deposited in duplicate on two glass slides pre-coated with 1% normal melting point agarose (Merck KGaA, cat. no. A4718), and covered with a 18 × 18 mm coverslip. Slides were placed for 5 min at 4 °C before coverslips were removed. Cell lysis was achieved by washing the slides with two successive lysis solutions at 4 °C, for 60 min each. The first lysis buffer contains 2.5 M NaCl, 100 mM EDTA, 10 mM Trizma base, 1% Triton X-100, and 10 mM DTT (pH 10), and the second lysis buffer consists of the first buffer with 0.05 mg/mL proteinase K.

For all the conditions, modified assay was also performed with extra step incubation with Formamidopyrimidine-DNA Glycosylase (Fpg), purchased from Professor Andrew Collins (University of Oslo, Norway), that convert oxidized purines into DNA single strand breaks. Enzyme diluted in buffer (30 µL) were applied in each gel, along with a coverslip, prior to incubation at 37 °C for 30 min in a humidified atmosphere (Azqueta et al. 2009).

Before electrophoresis, the DNA unwinding was promoted by incubating the slides in electrophoresis buffer (300 mM NaOH, 1 mM EDTA, pH 13.2), during 30 min at 4 °C. The electrophoresis tank (CSL-COM20, Cleaver Scientific Ltd; 31 × 34 × 9 cm) was always filled with 20 slides and 1.2 L of buffer, the necessary amount to cover slides only with a thin layer of buffer. Electrophoresis was conducted at 25 V (0.8 V/cm) for another 30 min, at 4 °C.

Finally, slides were neutralized in 1 × PBS (10 min, 4 °C), followed by 10-min distilled water, at 4 °C. The visualization and scoring of the comets were performed using fluorescent microscope, original magnification 200x), DAPI staining. The nucleoids were classified by visual scoring into 5 comet classes, according to the tail length and intensity from 0 (no tail) to 4 (almost all DNA in tail) (Collins 2004). The final score (expressed as ‘arbitrary units’ in a range of 0–400) was obtained by multiplying the mean percentage of nucleoids in each class by the corresponding factor, according to this formula: Genetic Damage Indicator (GDI) = [(% nucleoid class 0) × 0)] + [(% nucleoid class 1) × 1)] + [(% nucleoid class 2) × 2)] + [(% nucleoid class 3) × 3)] + [(% nucleoid class 4) × 4)]. The GDI and the parameter GDI_FPG were determined.

2.8. Statistical analysis

Descriptive statistics of data are presented as mean (M) and standard deviation (SD) when appropriate. Skewness and kurtosis coefficients were computed for univariate normality analysis purposes, and all values were within ± 2. To determine if the administration of MP and BP had a statistically significant effect on testis mitochondrial bioenergetics and antioxidant capacity in several organs, a multivariate analysis of variance (MANOVA) was performed, followed by a one-way analysis of variance (ANOVA) and post hoc tests, when appropriate (Hair et al. 2014). All statistical analysis was performed using SPSS (IBM SPSS Statistics 25). Statistically significant effects were assumed for p < 0.05.

3. Results

3.1. Animal weight, food and water consumption

There were no significant changes in feed or water intake, no significant changes in weight gain were observed (Table 1), and no mortality was observed during the experiment.

Table 1. Effect of treatment with two different concentrations of methyl and butylparaben on food and water consumption and percentage weight gain between the beginning of the experiment and three days after the last administration.

	Control	Methylparaben		Butylparaben
		100 mg/kg/day	200 mg/kg/day	100 mg/kg/day	200 mg/kg/day
Food consumption (g)	14.32 ± 2.08^a	13.97 ± 3.50^a	14.05 ± 1.29^a	13.33 ± 2.84^a	13.53 ± 2.31^a
Water consumption (ml)	30.53 ± 3.62^a	30.38 ± 2.88^a	30.50 ± 5.75^a	30.20 ± 2.61^a	28.68 ± 5.27^a
Weight gain (%)	31.28 ± 7.53^a	30.73 ± 6.42^a	29.34 ± 3.90^a	28.50 ± 3.41^a	29.18 ± 4.48^a

Paraben was subcutaneously administered in the inguinal zone at doses of 100 and 200 mg/kg (n = 6/group) daily to Wistar rats for 10 days. All results are given as mean ± SD of six animals. Different lowercase indicates groups that are statistically significantly different from each other (p < 0.05), whereas the same lowercase letter indicates no significant difference between groups (p > 0.05).

3.2. Tissue weight

Table 2 shows a decrease in the relative organ weight for all groups compared to the control group (CTRL), with the difference being statistically significant for the higher methylparaben concentration and both butylparaben concentrations (p < 0.005). The change in the relative weight of the seminal vesicles showed a pattern identical to that observed for the testicles, where only the Met-100 did not differ from the control (p = 0.5736).

Table 2. Effect of treatment with two different concentrations of methyl and butylparaben on the reproductive organ relative weight of rats.

	Control	Methylparaben		Butylparaben
		100 mg/kg/day	200 mg/kg/day	100 mg/kg/day	200 mg/kg/day
Right testicle (g)	0.74 ± 0.00^a	0.65 ± 0.00^a	0.60 ± 0.01^b	0.58 ± 0.03^b	0.55 ± 0.07^b
Left testicle (g)	0.69 ± 0.04^a	0.66 ± 0.00^a	0.61 ± 0.02^b	0.58 ± 0.01^b	0.55 ± 0.00^b
Seminal vesicles (g)	0.25 ± 0.03^a	0.21 ± 0.00^a	0.18 ± 0.01^b,c	0.16 ± 0.00^c	0.13 ± 0.00^d

All results are given as the mean ± SD of six animals. The relative organ weights (organ weight/animal weight) are multiplied by 100 for ease of presentation. Different lowercase indicates groups that are statistically significantly different from each other (p < 0.05), whereas the same lowercase letter indicates no significant difference between groups (p > 0.05).

3.3. Antioxidant enzyme activities

Figure 1(A, B, C and D) shows that methylparaben and butylparaben significantly affected antioxidant enzyme activities in testis compared to control. The superoxide dismutase activity increases in both groups treated with butylparaben, whereas in the case of methylparaben, only the Met-200 is statistically significant (p = 0.0136) (Figure 1A). As for catalase, there is an increase for both parabens in all tested concentrations. It is also worth mentioning that the But-200 group showed a significant increase when compared to either of the paraben-treated groups (p < 0.005) (Figure 1B). Glutathione reductase (Figure 1C) and glutathione S-transferase (Figure 1D) both undergo an inhibition in the activity induced by treatment with parabens, although the differences were only significant for butylparaben (p < 0.005).

Figure 1. Activity of oxidative stress enzymes in testes: (A) superoxide dismutase (SOD), (B) catalase (CAT), (C) glutathione reductase (GR), and (D) glutathione S-transferase (GST). Results are M ± SD of 8 independent experiments. The values marked with the same letter are not statistically significant between groups as determined by the Tukey’s post hoc test (p > 0.05).

3.4. Glutathione content and reducing power (GSH/GSSG)

Glutathione reducing power was established through GSH and GSSG levels and GSSG/GSH ratio. The reduced glutathione content did not show significant changes compared to the control group (Table 3). Oxidized glutathione, on the other hand, underwent a significant increase in the groups treated with butylparaben, when compared to the control group (Table 3). This difference in GSSG was reflected in the reducing capacity of glutathione (GSH/GSSG ratio) as the two groups treated with butylparaben show a significant decrease (p < 0.005) in the reducing capacity of glutathione when compared to the control group (Figure 2).

Figure 2. The glutathione (GSH)/glutathione disulfide (GSSG) ratio in rat testes. Results are M ± SD of 8 independent experiments. The values marked with the same letter are not statistically significant between groups as determined by the Tukey’s post hoc test (p > 0.05).

Table 3. Reduced glutathione (GSH) and oxidized glutathione (GSSG) content in the control group and groups treated with two different concentrations of methyl and butylparaben.

	Control	Methylparaben		Butylparaben
		100 mg/kg/day	200 mg/kg/day	100 mg/kg/day	200 mg/kg/day
GSH (nmol/mg)	4.07 ± 0.28	3.64 ± 0.27	3.73 ± 0.36	3.34 ± 0.38	3.39 ± 0.45
GSSG (nmol/mg)	1.53 ± 0.24^a	1.64 ± 0.37^a,b	1.95 ± 0.23^a,b	2.06 ± 0.18^b	2.15 ± 0.22^b

All values are given as mean ± SD of six animals. Different lowercase indicates groups that are statistically significantly different from each other (p < 0.05), whereas the same lowercase letter indicates no significant difference between groups (p > 0.05).

3.5. Lipid peroxidation (quantification of lipid hydroperoxides)

Treatment with butylparaben induced a significant increase in the concentration of hydroperoxides produced in testis when compared to the control and to the groups treated with methylparaben (p < 0.005), although this increase was not dependent on the dose administered (Figure 3).

Figure 3. Lipid hydroperoxide concentration in the testes of rats. Results are M ± SD of 8 independent experiments. The values marked with the same letter are not statistically significant between groups as determined by the Tukey’s post hoc test (p > 0.05).

3.6. DNA damage assessment (comet assay)

Analyzing GDI values (Figure 4), it was observed that all the treatments displayed higher values in comparison with the control. Nevertheless, butylparaben showed a significant increase compared with control and methylparaben groups. The detection of oxidized bases from the results after nucleoids were incubated with the DNA lesion-specific repair enzyme Fpg, indicated an increase in all groups. However, only the group But-100 showed a significant difference (p = 0.048). In Figure 5(A, B and C), we have comet assay images relating to stages 1, 2 and 3, which are representative of most of the cells observed in control (A), in But_100 (B) and But_200 (C).

Figure 4. Analysis of DNA damage. M ± SD of 4 independent experiments. DNA strand breaks, expressed as genetic damage index (GDI, grey), and DNA strand breaks plus oxidative damage, determined with formamidopyrimidine DNA glycosilase (Fpg) and expressed as GDI_Fpg (black), in rat testis cells. The values marked with the same letter are not statistically significant between groups as determined by the Tukey’s post hoc test (p > 0.05).

Figure 5. Comet assay images from rat sperm cells stained with DAPI, representative from control (almost cells class 1); But_100 (almost cells class 2) and But_200 Fpg (almost cells class 3).

4. Discussion

Parabens have been widely used as antimicrobial preservatives in processed foods and beverages, pharmaceuticals, and cosmetic products. Widespread human exposure to parabens has been extensively documented, and their safety has been questioned. It has been shown in some animal studies that parabens affect oxidative stress parameters, cause DNA damage and have adverse effects on male reproduction (Oishi 2002, Todorovac et al. 2021). However, the results remain controversial, as many of the studies are carried out under different conditions and in different models, which gives rise to contradictory results. Administration of methylparaben and butylparaben at concentrations of 100 and 200 mg/kg/day did not affect weight, feed, or water consumption (Table 1). This result was expected considering this animal species’ low observed effect level (LOEL) for butylparaben, which is 1600 mg/kg/day (Rodriguez et al. 1986). Despite this, the testicles significantly decreased in relative weight (p < 0.05) in the groups treated with butylparaben and the highest methylparaben concentration. These effects were more pronounced in the seminal vesicles, where the butylparaben shows a concentration-dependent effect (Table 2). This result agrees with another study in which the testis was also significantly smaller in all treated groups compared to controls (Oishi 2001). It was also previously demonstrated that the administration of methylparaben and butylparaben during pregnancy could decrease the mean relative weight of the testis and seminal vesicles in the F1 generation (Oliveira et al. 2020). A different result was obtained by Aydemir et al. (2019), in which 800 mg/kg/day did not affect the relative weight of the testis even though it increased that of the liver and kidney. However, the differences observed may result from the fact that the parabens were administered by oral gavage and not injected as in the present study. Previous studies have documented that several parabens can bind to androgen receptors exerting antiandrogenic activity (Satoh et al. 2005, Chen et al. 2007, Ding et al. 2017). Therefore, some of the above adverse effects (relative weight decrease on testis and seminal vesicles) may result from lowered circulating androgen action by parabens, which obstructs androgen signaling.

Superoxide dismutase and catalase are considered the first line of defense against the cell’s deleterious effects of oxygen radicals (Ighodaro and Akinloye 2018). Treatment with methyl and butyl parabens increased SOD and CAT activity (Figure 1A and B, respectively), with this increase being more expressive in groups treated with butylparaben. The ability of parabens to generate ROS and induce oxidative damage to DNA and mitochondria was related to the length of the alkyl chain (Samarasinghe et al. 2018, Martins et al. 2021), which can explain the differences observed between methyl and butyl parabens. Parabens’ effect on the oxidative stress enzymes is contradictory, a contradiction that can be explained by using different animal models and routes of administration (Lee et al. 2017, Aydemir et al. 2019, Schreiber et al. 2019). These differences make evident the need to define concerted approaches to assess the toxicity of this type of xenobiotics. Female albino mice of the Swiss strain, orally exposed to parabens, show a significant decrease in the activity of the most important enzymes from the antioxidant system (SOD, CAT, GR, GPx and GST) (Shah and Verma 2011). Another study carried out with young male Sprague-Dawley rats, subcutaneously treated with buthylparaben during one spermatogenic cycle, shows a decrease in CAT and GPx, and no differences in SOD. However, the GR activity was increased for 600 mg/Kg/day of butylparaben (Schreiber et al. 2019). Oral administration of butylparaben for 30 days has resulted in a marked increase in lipid peroxidation, which can be attributed to the decrease in non-enzymatic and enzymatic antioxidants (Shah and Verma 2011). In adipose tissue, methylparaben treatment was associated with lower glutathione reductase (GR) activity (Artacho-Cordón et al. 2019). In the present study, glutathione reductase and glutathione S-transferase (Figures 1C and D, respectively) significantly decreased in butylparaben groups (p < 0.05). This result is different from that observed by Schreiber et al. (2019), which may be explained by the difference in the experimental procedure. The lack of elevated GST activity would also contribute to DNA damage since GST prevents oxidative damage by conjugating a xenobiotic to GSH (Choi et al. 2008). The results reported in the literature regarding the changing oxidized and reduced glutathione content in animals treated with parabens are still inconclusive. In our study, GSH content did not change, although there was a significant increase in oxidized glutathione content (Table 3). This increase in GSSG causes a decrease in the GSH/GSSG ratio, which is significant (p < 0.05) for the two groups treated with butylparaben (Figure 2). It has been shown that exposure to butylparaben induces an increase in ROS, leading to cell damage (Samarasinghe et al. 2018, Ara et al. 2021). Methylparaben did not cause a significant increase in lipid peroxidation (p > 0.05), whereas butylparaben caused a significant increase (p < 0.05) even when compared to groups exposed to methylparaben (Figure 3). This result agrees with other studies indicating that the toxicological potential of parabens is related to the side chain’s length and the benzene ring’s presence (Engeli et al. 2017, Martins et al. 2021). It has already been demonstrated that parabens can affect the activity of some antioxidant enzymes and induce mitochondrial dysfunction, which is reflected in an imbalance between antioxidant species and ROS and consequently provokes oxidative damage.

The comet assay is an efficient tool to measure various kinds of DNA damage at the cellular level. Performing this protocol in rat testis cell suspensions, we showed differences for rats treated with butylparaben, with no effects observed with methylparaben (Figure 4). These data demonstrate that parabens’ genotoxic potential could depend on the side chain length. Indeed, the genotoxic effects of butylparaben have been demonstrated in human lymphocyte cultures and HepG2 cells (Bayülken and Tüylü 2019, Kizhedath et al. 2019). In an epithelial cell line derived from the Chinese hamster ovary, it was shown that after one hour of incubation with butylparaben (mM), a drastic migration of DNA was already observable by the comet test (Tayama et al. 2008). Jurewicz et al. (2017) observed a statistically significant positive association between urinary levels of butylparaben and human sperm DNA damage.

5. Conclusion

Paraben treatment increases SOD and CAT activity, which is not accompanied by GR and GST activity. This alteration in the enzymes of the antioxidant system does not allow it to provide antioxidant protection in the testis cells. These results suggest that the changes observed in the antioxidant system, mainly the decrease in the antioxidant capacity demonstrated by the GSH/GSSG ratio, led to oxidative damage and that it is likely responsible, in part, for the damage observed in the DNA. To the best of our knowledge, this is the first study to demonstrate the genotoxic effect of butylparaben on testicular cells when administered subcutaneously to rats.

Acknowledgements

We also want to thank Dr Malcolm Purves for kindly reviewing the English.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statements

https://www.dropbox.com/s/xxtsv001ecgunig/Data%20MP-BP.xlsx?dl=0.

Additional information

Funding

The authors thank FCT (Portugal’s Foundation for Science and Technology) for financial support through the CQ-VR Research Unit [UIDB/00616/2020, UIDP/00616/2020].