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Full Research Article

Oral Bisphenol A Exposure Induces Oxidative Stress and DNA-Damage Biomarkers and Alters Hepatic p53 in Rats: Modulatory Effects of Spirulina platensis

L
Lama A. Al-Otaibi1
P
Promy Virk1
M
Muath Q. Al-Ghadi1,*
https://orcid.org/0000-0002-0093-321X
1Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.

ABSTRACT

Background: Bisphenol A (BPA) is a widespread endocrine-disrupting environmental contaminant associated with oxidative and metabolic disturbances. This study investigated BPA-associated oxidative stress, serum 8-OHdG as a marker of oxidative DNA damage, hepatic p53 protein levels and selected hepatorenal biochemical markers in male rats and evaluated the modulatory effects of Spirulina platensis.

Methods: Sixty adult male Wistar rats were randomly allocated to six groups (n = 10/group): vehicle control, BPA at 120 mg/kg/day (BPA-LD), BPA at 240 mg/kg/day (BPA-HD), BPA-LD plus S. platensis (1000 mg/kg/day), BPA-HD plus S. platensis and S. platensis alone. Treatments were administered once daily by oral gavage for 21 consecutive days. Hepatic malondialdehyde (MDA), catalase (CAT), acyl-CoA oxidase 1 (ACOX1) and p53 protein levels, serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) and biochemical markers of liver and kidney function were evaluated.

Result: BPA exposure increased hepatic MDA and ACOX1 levels, serum 8-OHdG and hepatic p53 protein levels, with several responses being more pronounced at the lower BPA dose. Alterations were also observed in serum liver and kidney function markers. Co-administration of S. platensis attenuated several BPA-associated changes, including hepatic lipid peroxidation, serum 8-OHdG, increased hepatic ACOX1 and p53 protein levels and selected hepatorenal biochemical indices. However, the magnitude of the modulatory effect varied among the measured endpoints. These findings indicate that S. platensis may mitigate several biochemical consequences of BPA exposure, although further studies are required to clarify the underlying mechanisms.

INTRODUCTION

Endocrine-disrupting chemicals are pervasive environmental pollutants that can interfere with hormonal signaling and contribute to metabolic, developmental and reproductive disorders (La Merrill et al., 2020). Among these, bisphenol A (BPA) has raised particular concern because of its extensive use in polycarbonate plastics and epoxy resins and its presence in water, food-contact materials and household products (Manzoor et al., 2022). Human exposure to BPA is widespread, with BPA or its metabolites detected in biological samples including urine and serum (Rezg et al., 2014). Experimental studies have shown that BPA can induce oxidative stress, mitochondrial dysfunction, apoptosis and stress-responsive pathways, including p53, in multiple tissues (Abdulhameed et al., 2022; Nguyen et al., 2022; Wang et al., 2021).
       
Oral intake is a major route of BPA exposure. Following gastrointestinal absorption, BPA undergoes extensive hepatic first-pass glucuronidation and sulfation, making the liver a major site of BPA biotransformation and a relevant target for its toxicological effects (Manzoor et al., 2022; Hanioka et al., 2022). Hepatic BPA exposure has been associated with increased reactive oxygen species (ROS), impaired antioxidant defenses, lipid peroxidation and oxidative DNA damage, which may engage redox-sensitive cellular stress responses, including p53 (Uzunhisarcikli and Aslanturk, 2019). Because peroxisomal fatty acid oxidation can also contribute to cellular oxidant production, alterations in acyl-CoA oxidase 1 (ACOX1), the first enzyme of the peroxisomal β-oxidation pathway, may provide additional insight into BPA-associated disturbances in hepatic oxidative metabolism (Quijano et al., 2016). p53 was included as a complementary cellular stress-response marker because of its established involvement in responses to oxidative and DNA damage (Liu et al., 2020); however, changes in p53 protein abundance alone do not establish activation of downstream p53 signaling or apoptosis.
       
Spirulina platensis is a filamentous cyanobacterium widely consumed as a nutraceutical and reported to possess antioxidant, anti-inflammatory and immunomodulatory properties (Abdel-Daim et al., 2018; Finamore et al., 2017). Spirulina-derived compounds have also shown protective effects against BPA-associated toxicity, including nephrotoxicity, primarily through antioxidant and anti-inflammatory actions (Chouala et al., 2024).
       
Although the antioxidant effects of S. platensis and the oxidative toxicity of BPA have been investigated separately, integrated in vivo evidence examining oxidative stress, circulating 8-OHdG, hepatic ACOX1 and p53 protein responses and hepatorenal biochemical alterations within the same BPA exposure model remains limited. The present study therefore integrates these endpoints to characterize BPA-associated responses across oxidative, metabolic, stress-response and hepatorenal domains and to determine the extent to which S. platensis modulates these alterations.
       
We hypothesized that repeated oral BPA exposure would induce oxidative stress, increase serum 8-OHdG as a circulating marker of  oxidative DNA damage and alter hepatic ACOX1 and p53 protein levels, whereas S. platensis co-treatment would attenuate these changes. Because the BPA doses used in this study were supra-environmental, the experimental model was intended to characterize biological responses under short-term toxicological exposure rather than reproduce typical human environmental exposure (EFSA CEP Panel, 2023). Therefore, the present study aimed to evaluate the effects of two oral BPA doses on oxidative stress, serum 8-OHdG, hepatic ACOX1 and p53 protein levels and selected hepatorenal biochemical markers in male Wistar rats and to determine whether co-administration of S. platensis could attenuate these BPA-associated alterations.

MATERIALS AND METHODS

Chemicals and reagents
 
Analytical-grade bisphenol A (BPA; CAS No. 80-05-7; Cat. No. B0494; >99% purity) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and dissolved in corn oil for oral administration. Certified organic Spirulina platensis powder (California Gold Nutrition®, Perris, CA, USA) was freshly suspended in distilled water at a concentration of 150 mg/mL for oral administration. Commercial assay kits were used to evaluate oxidative stress and cellular-response biomarkers. Catalase (CAT) activity and malondialdehyde (MDA) levels were measured using colorimetric assay kits from Abcam (Cambridge, UK; Cat. Nos. ab83464 and ab118970, respectively). The CAT and MDA assays had analytical ranges of 0-10 nmol/well and 0-20 nmol, respectively. Serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) was quantified using a competitive DNA Damage ELISA Kit (Enzo Life Sciences, Farmingdale, NY, USA; Cat. No. ADI-EKS-350), with a standard range of 0.94-60 ng/mL, a sensitivity of 0.59 ng/mL and intra- and inter-assay coefficients of variation (CVs) below 10%. Hepatic acyl-CoA oxidase 1 (ACOX1) was measured using a quantitative sandwich ELISA kit (MyBioSource, Inc., San Diego, CA, USA; Cat. No. MBS9325630), with a detection range of 0.5-16 ng/mL, a sensitivity of 0.1 ng/mL and intra- and inter-assay CVs below 15%. Hepatic p53 protein was measured using a sandwich ELISA kit (MyBioSource, Inc.; Cat. No. MBS723886), with a standard range of 0-50 ng/mL, a sensitivity of 0.1 ng/mL and same-lot and different-lot CVs below 10% and 12%, respectively.
 
Animals and ethical approval
 
Sixty adult male Wistar rats with an initial body weight of 150±10 g were obtained from the Animal House Facility at King Saud University, Riyadh, Saudi Arabia. Animals were acclimated for 7 days before the experiment and maintained under controlled environmental conditions (22±2°C; 12 h light/12 h dark cycle), with ad libitum access to a standard pellet diet and water.
       
All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at King Saud University (Approval No. KSU-SE-25-38) and were conducted in accordance with the approved institutional guidelines for the care and use of laboratory animals.
 
Experimental design and treatments
 
Following the 7-day acclimation period, the 60 rats were assigned to six experimental groups (n = 10 per group) using simple randomization. The group size was selected based on previous comparable toxicological studies in Wistar rats (Atasever et al., 2020) and established guidance for repeated-dose toxicity studies (OECD, 2008). The groups were: (1) control, receiving distilled water; (2) BPA-LD, receiving BPA at 120 mg/kg body weight/day; (3) BPA-HD, receiving BPA at 240 mg/kg body weight/day; (4) BPA-LD + S. platensis, receiving BPA at 120 mg/kg body weight/day plus S. platensis at 1000 mg/kg body weight/day; (5) BPA-HD + S. platensis, receiving BPA at 240 mg/kg body weight/day plus S. platensis at 1000 mg/kg body weight/day and (6) S. platensis-only, receiving S. platensis at 1000 mg/kg body weight/day.
       
All treatments were administered once daily by oral gavage for 21 consecutive days. BPA was dissolved in corn oil, whereas S. platensis was suspended in distilled water. The gavage volume was 5 mL/kg body weight and was adjusted according to individual body weight (OECD, 2008). The control group received distilled water by oral gavage. In the combination groups, BPA was administered first, followed by S. platensis 15 min later. Biochemical outcome assessments were performed by laboratory personnel blinded to group allocation. No animals or samples were excluded from the study or subsequent analyses.
       
The BPA doses were selected based on a previous toxicological study using oral doses of 120 and 240 mg/kg body weight/day (Samova et al., 2018), whereas the S. platensis dose was selected based on previous experimental studies in Wistar rats (Mohan et al., 2006; Seyidoglu et al., 2020). The BPA doses were supra-environmental and were selected to characterize biological responses during short-term toxicological exposure rather than to reproduce typical human environmental exposure.
 
Sample collection
 
At the end of the 21-day treatment period, animals were fasted for approximately 12 h and euthanized by carbon dioxide (CO2) inhalation at a displacement rate of 30% of chamber volume per minute in accordance with animal welfare guidelines (American Veterinary Medical Association, 2020). Blood was collected by cardiac puncture, allowed to clot and centrifuged at 3000 rpm (approximately 1000 × g) for 10 min to obtain serum. Serum samples were stored at -80°C until analysis.
       
Liver tissues were excised, rinsed with ice-cold physiological saline (0.9% NaCl), snap-frozen in liquid nitrogen and stored at -80°C until biochemical analysis.
 
Biochemical and protein biomarker assays
 
Hepatic CAT activity and MDA levels were determined using colorimetric assays, whereas hepatic ACOX1 and p53 protein levels and serum 8-OHdG were quantified using the respective ELISA kits described above, according to the manufacturers’ instructions. Liver tissues were prepared separately for each hepatic endpoint using the tissue-to-buffer ratios specified for the respective assays.
       
For CAT activity, 100 mg of liver tissue was homogenized in 200 µL of ice-cold assay buffer. The homogenate was processed according to the manufacturer’s protocol and the resulting supernatant was used for the assay. CAT activity was determined colorimetrically at 570 nm using a microplate reader and expressed as nmol/min/mL.
       
For MDA measurement, 10 mg of liver tissue was homogenized in 300 µL of MDA Lysis Buffer supplemented with 3 µL of BHT. The homogenate was processed according to the manufacturer’s protocol and MDA levels were determined colorimetrically at 532 nm based on the formation of an MDA–thiobarbituric acid (TBA) adduct. Results were expressed as nmol/mg tissue.
       
For ACOX1 measurement, 10 mg of liver tissue was homogenized in 100 µL of phosphate-buffered saline (PBS). The homogenate was processed according to the manufacturer’s protocol and the resulting supernatant was analyzed using the quantitative sandwich ELISA. Absorbance was measured at 450 nm using a microplate reader and ACOX1 concentrations were determined from the corresponding standard curve and expressed as ng/mL of liver-homogenate supernatant.
       
For p53 measurement, 500 mg of liver tissue was homogenized in 500 µL of PBS. The homogenate was processed according to the manufacturer’s protocol and the resulting supernatant was analyzed using the sandwich ELISA. Absorbance was measured at 450 nm using a microplate reader and p53 concentrations were determined from the corresponding standard curve and expressed as ng/mL of liver-homogenate supernatant.
       
For 8-OHdG measurement, serum samples were diluted 1:20 (v/v) in the supplied sample diluent and analyzed using the competitive ELISA according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a microplate reader and 8-OHdG concentrations were determined from the standard curve, corrected for the dilution factor and expressed as pg/mL.
       
Total protein concentration was not determined; therefore, CAT, ACOX1 and p53 values were reported in the assay-specific units generated according to the respective kit procedures rather than normalized to total protein. MDA levels were expressed as nmol/mg tissue based on the original tissue mass used for the assay. 
 
Liver and kidney function tests
 
Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and urea and creatinine concentrations were measured using an AU680 clinical chemistry analyzer (Beckman Coulter, Inc., Brea, CA, USA) with the manufacturer’s reagents and according to the manufacturer’s protocols.
 
Statistical analysis
 
Statistical analyses were performed using IBM SPSS Statistics, version 29.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean±standard error of the mean (SEM). Differences among the six experimental groups were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons. The assumptions of normality and homogeneity of variances were assessed using the Shapiro-Wilk and Levene’s tests, respectively. Statistical significance was set at P < 0.05. ANOVA results are reported as F statistics with the corresponding degrees of freedom and exact P values where available. All 10 animals per group were included in the analysis of each outcome, with no exclusions. Numerical mean ± SEM values for the measured outcomes are provided in Table 1. In the graphical presentation of the results, groups not sharing a common superscript letter were considered significantly different according to Tukey’s HSD test.

Table 1: Numerical summary of measured biochemical and protein endpoints across experimental groups.

RESULTS AND DISCUSSION

Oxidative stress, oxidative DNA damage and ACOX1 response
 
Hepatic MDA levels
 
Hepatic MDA levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 39.179, P<0.001).  MDA levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with no significant difference between the two BPA-treated groups. Spirulina platensis co-treatment significantly reduced MDA levels in both HD+S and LD+S groups to values that did not differ significantly from the control. The S. platensis-only group also did not differ significantly from the control (Fig 1; Table 1).

Fig 1: Hepatic malondialdehyde (MDA) levels.


 
Hepatic catalase activity
 
Hepatic CAT activity differed significantly among experimental groups (one-way ANOVA: F(5,54) = 190.596, P<0.001). CAT activity was significantly increased in the BPA-LD group compared with the control (P<0.05), whereas the BPA-HD group did not differ significantly from the control. S. platensis co-treatment significantly reduced CAT activity in the LD+S group compared with BPA-LD, although it remained significantly higher than the control. CAT activity in the HD+S and S. platensis-only groups did not differ significantly from the control (Fig 2; Table 1).

Fig 2: Hepatic catalase (CAT) activity.



Serum 8-OHdG levels
 
Serum 8-OHdG, assessed as a circulating marker of oxidative DNA damage, differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 61.893, P<0.001). Serum 8-OHdG levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with significantly higher levels in BPA-LD than BPA-HD. S. platensis co-treatment significantly reduced 8-OHdG levels in both HD+S and LD+S groups compared with their respective BPA-only groups; however, both co-treatment groups remained significantly higher than the control. The S. platensis-only group did not differ significantly from the control (Fig 3; Table 1).

Fig 3: Serum 8-hydroxy-2¢-deoxyguanosine (8-OHdG) levels.


 
Hepatic ACOX1 levels
 
Hepatic ACOX1 levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 224.280, P<0.001). ACOX1 levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with no significant difference between the two BPA-treated groups. S. platensis co-treatment significantly reduced ACOX1 levels in both HD+S and LD+S groups compared with their respective BPA-only groups. ACOX1 levels in HD+S did not differ significantly from the control, whereas LD+S remained significantly higher. The S. platensis-only group did not differ significantly from the control (Fig 4; Table 1).

Fig 4: Hepatic acyl-CoA oxidase 1 (ACOX1) levels.


 
Hepatic p53 protein levels
 
Hepatic p53 protein levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 195.916, P<0.001). p53 protein levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with no significant difference between the two BPA-treated groups. S. platensis co-treatment significantly reduced p53 levels in both HD+S and LD+S groups compared with their respective BPA-only groups. Both co-treatment groups showed significantly lower p53 levels than the control and differed significantly from each other. The S. platensis-only group did not differ significantly from the control (Fig 5; Table 1).

Fig 5: Hepatic p53 protein levels.


 
Liver and kidney function markers
 
Serum urea levels
 
Serum urea levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 38.979, P<0.001). Urea levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with significantly higher levels in BPA-LD than BPA-HD. S. platensis co-treatment was associated with lower mean urea levels in both co-treatment groups. Urea levels in HD+S did not differ significantly from BPA-HD but remained significantly higher than the control, whereas LD+S was significantly lower than BPA-LD and did not differ significantly from either the control or BPA-HD. The S. platensis- only group did not differ significantly from the control (Fig 6; Table 1).

Fig 6: Serum urea levels.


 
Serum creatinine levels
 
Serum creatinine levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 8.745, P = 0.001). Creatinine was significantly increased in the BPA-LD group compared with the control (P<0.05), whereas BPA-HD did not differ significantly from the control. S. platensis co-treatment significantly reduced creatinine in the LD+S group compared with BPA-LD, to a level that did not differ significantly from the control. Creatinine levels in the HD+S and S. platensis-only groups also did not differ significantly from the control (Fig 7; Table 1).

Fig 7: Serum creatinine levels.


 
Serum ALT and AST activities
 
Serum ALT activity differed significantly among experimental groups (one-way ANOVA: F(5,54) = 112.523, P<0.001). ALT activity was significantly increased in the BPA-LD group compared with the control and all other experimental groups (P < 0.05), whereas BPA-HD, HD+S, LD+S and S. platensis-only groups did not differ significantly from the control. S. platensis co-treatment significantly reduced ALT activity in the LD+S group compared with the corresponding BPA-LD group (Fig 8; Table 1).

Fig 8: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities.


       
Serum AST activity differed significantly among experimental groups (one-way ANOVA: F(5,54) = 27.186, P<0.001).  AST activity was significantly increased in the BPA-HD, BPA-LD, HD+S and LD+S groups compared with the control (P<0.05), with BPA-LD showing significantly higher activity than all other experimental groups. S. platensis co-treatment significantly reduced AST activity in the LD+S group compared with BPA-LD, whereas HD+S did not differ significantly from BPA-HD. The S. platensis-only group did not differ significantly from the control (Fig 8; Table 1).
       
The present study shows that repeated oral exposure to BPA induces multiple biochemical alterations involving oxidative stress, circulating 8-OHdG, hepatic ACOX1 and p53 protein levels and markers of hepatic and renal function in male rats. These effects were accompanied by increased hepatic MDA and ACOX1 levels, differential changes in CAT activity, elevated circulating 8-OHdG and changes in serum ALT, AST, urea and creatinine. Co-administration of Spirulina platensis attenuated several of these responses, although  the degree of modulation varied according to the measured endpoint and BPA dose. Collectively, these findings support oxidative stress and altered circulating 8-OHdG as prominent biochemical responses to BPA exposure and indicate that S. platensis can modulate several biochemical responses associated with BPA exposure.
       
One of the clearest effects of BPA exposure was the increase in hepatic MDA levels at both doses. MDA is widely used as an indicator of lipid peroxidation and reflects oxidative injury to cellular membranes. The elevation observed in the present study is consistent with previous experimental evidence showing that BPA can disturb cellular redox homeostasis and promote oxidative stress in different tissues (Manzoor et al., 2022; Rezg et al., 2014; Abdulhameed et al., 2022; Boudali et al., 2026; Ravikumar et al., 2022). Increased lipid peroxidation may result from excessive generation of reactive oxygen species together with an insufficient antioxidant response, ultimately affecting membrane integrity and cellular function. Importantly, co-treatment with S. platensis reduced MDA to levels that were not significantly different from the control, indicating attenuation of the BPA-associated increase in lipid peroxidation.
       
The CAT response was more complex. CAT activity was markedly increased in the BPA-LD group, whereas the BPA-HD group did not differ significantly from the control. This response indicates that antioxidant enzyme activity did not increase proportionally with BPA dose. Increased CAT activity at the lower BPA dose may represent an adaptive response to enhanced hydrogen peroxide production, whereas the absence of a comparable increase at the higher dose may reflect a different balance between oxidative challenge and antioxidant capacity. Nevertheless, the present data do not establish the mechanism responsible for this pattern and this interpretation should therefore remain cautious.
       
The increase in serum 8-OHdG is consistent with enhanced oxidative DNA damage; however, as a circulating biomarker, it does not identify the tissue source of DNA oxidation. Both BPA doses significantly increased 8-OHdG, with a greater response in the BPA-LD group than in the BPA-HD group. 8-OHdG is generated following oxidative modification of guanine residues and is commonly used as a marker of oxidative DNA damage. The parallel elevation of hepatic MDA and circulating 8-OHdG therefore suggests that BPA exposure was associated with both lipid peroxidation and systemic oxidative DNA damage. S. platensis significantly reduced 8-OHdG in both co-treatment groups; however, levels remained significantly higher than those of the control. Thus, unlike the near normalization observed for MDA, the modulatory effect of S. platensis on circulating 8-OHdG was only partial.
       
Hepatic ACOX1 levels were also increased following exposure to both BPA doses. ACOX1 catalyzes the first and rate-limiting step of peroxisomal fatty-acid β-oxidation, a process that generates hydrogen peroxide as a metabolic by-product. Increased ACOX1 may therefore contribute to the altered hepatic oxidative environment observed following BPA exposure (Quijano et al., 2016). Co-administration of S. platensis markedly attenuated this response. ACOX1 in the HD+S group was comparable to the control, whereas the LD+S group remained significantly elevated. The differential responses of the two co-treatment groups further illustrate the endpoint-specific nature of the effects observed.
       
A particularly relevant finding was the increase in hepatic p53 protein levels following BPA exposure. p53 is a central cellular stress-response protein that participates in the regulation of DNA-damage responses, cell-cycle control, DNA repair and apoptosis (Liu et al., 2020). The simultaneous increases in circulating 8-OHdG and hepatic p53 protein abundance observed in the BPA-treated groups are therefore consistent with cellular stress responses associated with oxidative condition.
       
Co-administration of S. platensis significantly reduced hepatic p53 levels in both BPA-treated groups. Interestingly, p53 levels in the co-treatment groups were lower than those in the control group rather than merely returning to control values. Therefore, this response should not be interpreted as simple normalization of p53. Instead, it indicates substantial modulation of the hepatic p53 response during combined BPA and S. platensis exposure. Because downstream components of the p53 pathway were not assessed, the biological significance of this reduction cannot be established from the present data alone.
       
The modulatory responses observed with S. platensis are consistent with its reported antioxidant properties. S. platensis contains several bioactive constituents, including phycocyanin, carotenoids, phenolic compounds, vitamins and other antioxidant components that may contribute to its ability to limit oxidative injury (Seyidoglu et al., 2020; Abdel-Daim et al., 2018; Finamore et al., 2017; Chouala et al., 2024). Previous experimental studies have similarly reported protective effects of S. platensis against chemically induced oxidative stress and tissue injury (Abdel-Daim et al., 2018; Finamore et al., 2017). Of particular relevance, Chouala et al., (2024) reported protection by Arthrospira platensis against BPA-induced nephrotoxicity in rats, providing relevant support for its potential to modulate BPA-associated toxicity.
       
Changes in serum biochemical markers further indicated that BPA exposure was associated with biochemical alterations in organ-function markers beyond the oxidative stress endpoints. The BPA-LD group showed particularly pronounced elevations in ALT and AST, whereas the BPA-HD group exhibited a significant increase in AST without a corresponding significant increase in ALT. Similar associations between chemically induced hepatic oxidative stress, lipid peroxidation and alterations in serum liver enzymes have been reported in experimental rat models (Ayaz et al., 2017). Similarly, serum urea increased following both BPA doses, while creatinine was significantly elevated only in the BPA-LD group. Co-administration of S. platensis attenuated several of these alterations, particularly in the BPA-LD group, although the extent of attenuation varied among biomarkers.
       
The apparently stronger responses to the lower BPA dose for several endpoints, including CAT, 8-OHdG, ALT, AST, urea and creatinine, warrant careful interpretation. BPA has been reported to produce non-monotonic dose-response patterns under some experimental conditions (Vandenberg et al., 2012; Vandenberg, 2014; Welshons et al., 2003). However, the present experiment was not specifically designed to characterize the shape or mechanism of a non-monotonic dose-response relationship. Accordingly, the observed differences between the two BPA doses should be regarded as endpoint-specific responses rather than definitive evidence of a particular dose-response mechanism.
       
It is also important to place the doses used in the present study in the appropriate toxicological context. The BPA doses of 120 and 240 mg/kg/day are substantially higher than typical human environmental exposures and were selected based on previous toxicological work using these dose levels (Samova et al., 2018). Consequently, the findings should not be directly extrapolated to expected effects at ordinary human exposure levels. Rather, the study provides experimental evidence regarding biological responses to short-term, high-dose BPA exposure and the extent to which S. platensis modifies those responses.
       
Several limitations should be considered. First, only male rats were examined, limiting extrapolation to females or other biological contexts. Second, the 21-day exposure period represents a relatively short toxicological model and does not address chronic exposure. Third, the BPA doses were supra-environmental and therefore do not reproduce typical human exposure conditions. Fourth, the mechanistic assessment was limited to selected markers of oxidative stress, peroxisomal metabolism and p53 protein abundance. Histopathological evaluation, direct tissue-based assays and additional molecular markers of DNA repair, apoptosis, inflammation and antioxidant signaling would provide a more comprehensive characterization of tissue response. In addition, serum 8-OHdG is a systemic biomarker and does not localize DNA oxidation to hepatic tissue, while increased p53 protein abundance alone does not establish p53-pathway activation or apoptosis. Finally, the study demonstrates biochemical associations but cannot establish direct causal relationships among ACOX1 levels, oxidative stress, DNA damage and p53 signaling.

CONCLUSION

Repeated oral exposure to BPA induced oxidative stress, elevated serum 8-OHdG consistent with oxidative DNA damage, altered hepatic p53 protein levels and changes in liver and kidney function markers in male rats. Co-administration of Spirulina platensis attenuated several BPA-associated biochemical changes, particularly hepatic lipid peroxidation and serum 8-OHdG, increased hepatic ACOX1 levels, the BPA-associated increase in hepatic p53 protein levels and selected biochemical indices of hepatic and renal function. However, the magnitude of modulation varied among endpoints and several responses did not increase proportionally with BPA dose. These findings indicate that S. platensis modulates multiple biochemical responses associated with BPA exposure in this experimental model, while highlighting the need for further studies using environmentally relevant BPA doses, longer exposure periods, histopathological assessment and broader molecular analyses to clarify the mechanisms underlying these modulatory effects.

ACKNOWLEDGEMENT

The authors extend their appreciation to the Ongoing Research Funding Program (ORF-2026-811), King Saud University, Riyadh, Saudi Arabia, for funding this work. The authors also thank the staff of the Animal House Facility, Department of Zoology, King Saud University, for their technical support and assistance with animal care.
 
Funding
 
This work was supported by the Ongoing Research Funding Program (ORF-2026-811), King Saud University, Riyadh, Saudi Arabia.
 
Animal ethics
 
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at King Saud University, Riyadh, Saudi Arabia (Approval No. KSU-SE-25-38). All experimental procedures were conducted in accordance with the approved institutional guidelines for the care and use of laboratory animals.
 
Author contributions
 
Lama A. Al-Otaibi and Muath Q. Al-Ghadi conceived the study. Lama A. Al-Otaibi and Promy Virk contributed to the methodology. Lama A. Al-Otaibi performed the experiments, conducted the laboratory procedures and collected and curated the data. Promy Virk contributed to validation and study supervision. Muath Q. Al-Ghadi supervised the research and administered the project. Lama A. Al-Otaibi prepared the original draft of the manuscript. Promy Virk and Muath Q. Al-Ghadi reviewed and revised the manuscript for important intellectual content. All authors read and approved the final version of the manuscript.
 
Data availability statement
 
The data supporting the findings of this study are available from the corresponding author upon reasonable request.

CONFLICT OF INTEREST

On behalf of all co-authors, we confirm that the authors declare no conflicts of interest, financial or otherwise, that could have influenced the research presented in the manuscript.

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    Oral Bisphenol A Exposure Induces Oxidative Stress and DNA-Damage Biomarkers and Alters Hepatic p53 in Rats: Modulatory Effects of Spirulina platensis

    L
    Lama A. Al-Otaibi1
    P
    Promy Virk1
    M
    Muath Q. Al-Ghadi1,*
    https://orcid.org/0000-0002-0093-321X
    1Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.

    ABSTRACT

    Background: Bisphenol A (BPA) is a widespread endocrine-disrupting environmental contaminant associated with oxidative and metabolic disturbances. This study investigated BPA-associated oxidative stress, serum 8-OHdG as a marker of oxidative DNA damage, hepatic p53 protein levels and selected hepatorenal biochemical markers in male rats and evaluated the modulatory effects of Spirulina platensis.

    Methods: Sixty adult male Wistar rats were randomly allocated to six groups (n = 10/group): vehicle control, BPA at 120 mg/kg/day (BPA-LD), BPA at 240 mg/kg/day (BPA-HD), BPA-LD plus S. platensis (1000 mg/kg/day), BPA-HD plus S. platensis and S. platensis alone. Treatments were administered once daily by oral gavage for 21 consecutive days. Hepatic malondialdehyde (MDA), catalase (CAT), acyl-CoA oxidase 1 (ACOX1) and p53 protein levels, serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) and biochemical markers of liver and kidney function were evaluated.

    Result: BPA exposure increased hepatic MDA and ACOX1 levels, serum 8-OHdG and hepatic p53 protein levels, with several responses being more pronounced at the lower BPA dose. Alterations were also observed in serum liver and kidney function markers. Co-administration of S. platensis attenuated several BPA-associated changes, including hepatic lipid peroxidation, serum 8-OHdG, increased hepatic ACOX1 and p53 protein levels and selected hepatorenal biochemical indices. However, the magnitude of the modulatory effect varied among the measured endpoints. These findings indicate that S. platensis may mitigate several biochemical consequences of BPA exposure, although further studies are required to clarify the underlying mechanisms.

    INTRODUCTION

    Endocrine-disrupting chemicals are pervasive environmental pollutants that can interfere with hormonal signaling and contribute to metabolic, developmental and reproductive disorders (La Merrill et al., 2020). Among these, bisphenol A (BPA) has raised particular concern because of its extensive use in polycarbonate plastics and epoxy resins and its presence in water, food-contact materials and household products (Manzoor et al., 2022). Human exposure to BPA is widespread, with BPA or its metabolites detected in biological samples including urine and serum (Rezg et al., 2014). Experimental studies have shown that BPA can induce oxidative stress, mitochondrial dysfunction, apoptosis and stress-responsive pathways, including p53, in multiple tissues (Abdulhameed et al., 2022; Nguyen et al., 2022; Wang et al., 2021).
           
    Oral intake is a major route of BPA exposure. Following gastrointestinal absorption, BPA undergoes extensive hepatic first-pass glucuronidation and sulfation, making the liver a major site of BPA biotransformation and a relevant target for its toxicological effects (Manzoor et al., 2022; Hanioka et al., 2022). Hepatic BPA exposure has been associated with increased reactive oxygen species (ROS), impaired antioxidant defenses, lipid peroxidation and oxidative DNA damage, which may engage redox-sensitive cellular stress responses, including p53 (Uzunhisarcikli and Aslanturk, 2019). Because peroxisomal fatty acid oxidation can also contribute to cellular oxidant production, alterations in acyl-CoA oxidase 1 (ACOX1), the first enzyme of the peroxisomal β-oxidation pathway, may provide additional insight into BPA-associated disturbances in hepatic oxidative metabolism (Quijano et al., 2016). p53 was included as a complementary cellular stress-response marker because of its established involvement in responses to oxidative and DNA damage (Liu et al., 2020); however, changes in p53 protein abundance alone do not establish activation of downstream p53 signaling or apoptosis.
           
    Spirulina platensis     is a filamentous cyanobacterium widely consumed as a nutraceutical and reported to possess antioxidant, anti-inflammatory and immunomodulatory properties (Abdel-Daim et al., 2018; Finamore et al., 2017). Spirulina-derived compounds have also shown protective effects against BPA-associated toxicity, including nephrotoxicity, primarily through antioxidant and anti-inflammatory actions (Chouala et al., 2024).
           
    Although the antioxidant effects of S. platensis and the oxidative toxicity of BPA have been investigated separately, integrated in vivo evidence examining oxidative stress, circulating 8-OHdG, hepatic ACOX1 and p53 protein responses and hepatorenal biochemical alterations within the same BPA exposure model remains limited. The present study therefore integrates these endpoints to characterize BPA-associated responses across oxidative, metabolic, stress-response and hepatorenal domains and to determine the extent to which S. platensis modulates these alterations.
           
    We hypothesized that repeated oral BPA exposure would induce oxidative stress, increase serum 8-OHdG as a circulating marker of  oxidative DNA damage and alter hepatic ACOX1 and p53 protein levels, whereas S. platensis co-treatment would attenuate these changes. Because the BPA doses used in this study were supra-environmental, the experimental model was intended to characterize biological responses under short-term toxicological exposure rather than reproduce typical human environmental exposure (EFSA CEP Panel, 2023). Therefore, the present study aimed to evaluate the effects of two oral BPA doses on oxidative stress, serum 8-OHdG, hepatic ACOX1 and p53 protein levels and selected hepatorenal biochemical markers in male Wistar rats and to determine whether co-administration of S. platensis could attenuate these BPA-associated alterations.

    MATERIALS AND METHODS

    Chemicals and reagents
     
    Analytical-grade bisphenol A (BPA; CAS No. 80-05-7; Cat. No. B0494; >99% purity) was obtained from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and dissolved in corn oil for oral administration. Certified organic Spirulina platensis powder (California Gold Nutrition®, Perris, CA, USA) was freshly suspended in distilled water at a concentration of 150 mg/mL for oral administration. Commercial assay kits were used to evaluate oxidative stress and cellular-response biomarkers. Catalase (CAT) activity and malondialdehyde (MDA) levels were measured using colorimetric assay kits from Abcam (Cambridge, UK; Cat. Nos. ab83464 and ab118970, respectively). The CAT and MDA assays had analytical ranges of 0-10 nmol/well and 0-20 nmol, respectively. Serum 8-hydroxy-2′-deoxyguanosine (8-OHdG) was quantified using a competitive DNA Damage ELISA Kit (Enzo Life Sciences, Farmingdale, NY, USA; Cat. No. ADI-EKS-350), with a standard range of 0.94-60 ng/mL, a sensitivity of 0.59 ng/mL and intra- and inter-assay coefficients of variation (CVs) below 10%. Hepatic acyl-CoA oxidase 1 (ACOX1) was measured using a quantitative sandwich ELISA kit (MyBioSource, Inc., San Diego, CA, USA; Cat. No. MBS9325630), with a detection range of 0.5-16 ng/mL, a sensitivity of 0.1 ng/mL and intra- and inter-assay CVs below 15%. Hepatic p53 protein was measured using a sandwich ELISA kit (MyBioSource, Inc.; Cat. No. MBS723886), with a standard range of 0-50 ng/mL, a sensitivity of 0.1 ng/mL and same-lot and different-lot CVs below 10% and 12%, respectively.
     
    Animals and ethical approval
     
    Sixty adult male Wistar rats with an initial body weight of 150±10 g were obtained from the Animal House Facility at King Saud University, Riyadh, Saudi Arabia. Animals were acclimated for 7 days before the experiment and maintained under controlled environmental conditions (22±2°C; 12 h light/12 h dark cycle), with ad libitum access to a standard pellet diet and water.
           
    All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) at King Saud University (Approval No. KSU-SE-25-38) and were conducted in accordance with the approved institutional guidelines for the care and use of laboratory animals.
     
    Experimental design and treatments
     
    Following the 7-day acclimation period, the 60 rats were assigned to six experimental groups (n = 10 per group) using simple randomization. The group size was selected based on previous comparable toxicological studies in Wistar rats (Atasever et al., 2020) and established guidance for repeated-dose toxicity studies (OECD, 2008). The groups were: (1) control, receiving distilled water; (2) BPA-LD, receiving BPA at 120 mg/kg body weight/day; (3) BPA-HD, receiving BPA at 240 mg/kg body weight/day; (4) BPA-LD + S. platensis, receiving BPA at 120 mg/kg body weight/day plus S. platensis at 1000 mg/kg body weight/day; (5) BPA-HD + S. platensis, receiving BPA at 240 mg/kg body weight/day plus S. platensis at 1000 mg/kg body weight/day and (6) S. platensis-only, receiving S. platensis at 1000 mg/kg body weight/day.
           
    All treatments were administered once daily by oral gavage for 21 consecutive days. BPA was dissolved in corn oil, whereas S. platensis was suspended in distilled water. The gavage volume was 5 mL/kg body weight and was adjusted according to individual body weight (OECD, 2008). The control group received distilled water by oral gavage. In the combination groups, BPA was administered first, followed by S. platensis 15 min later. Biochemical outcome assessments were performed by laboratory personnel blinded to group allocation. No animals or samples were excluded from the study or subsequent analyses.
           
    The BPA doses were selected based on a previous toxicological study using oral doses of 120 and 240 mg/kg body weight/day (Samova et al., 2018), whereas the S. platensis dose was selected based on previous experimental studies in Wistar rats (Mohan et al., 2006; Seyidoglu et al., 2020). The BPA doses were supra-environmental and were selected to characterize biological responses during short-term toxicological exposure rather than to reproduce typical human environmental exposure.
     
    Sample collection
     
    At the end of the 21-day treatment period, animals were fasted for approximately 12 h and euthanized by carbon dioxide (CO2) inhalation at a displacement rate of 30% of chamber volume per minute in accordance with animal welfare guidelines (American Veterinary Medical Association, 2020). Blood was collected by cardiac puncture, allowed to clot and centrifuged at 3000 rpm (approximately 1000 × g) for 10 min to obtain serum. Serum samples were stored at -80°C until analysis.
           
    Liver tissues were excised, rinsed with ice-cold physiological saline (0.9% NaCl), snap-frozen in liquid nitrogen and stored at -80°C until biochemical analysis.
     
    Biochemical and protein biomarker assays
     
    Hepatic CAT activity and MDA levels were determined using colorimetric assays, whereas hepatic ACOX1 and p53 protein levels and serum 8-OHdG were quantified using the respective ELISA kits described above, according to the manufacturers’ instructions. Liver tissues were prepared separately for each hepatic endpoint using the tissue-to-buffer ratios specified for the respective assays.
           
    For CAT activity, 100 mg of liver tissue was homogenized in 200 µL of ice-cold assay buffer. The homogenate was processed according to the manufacturer’s protocol and the resulting supernatant was used for the assay. CAT activity was determined colorimetrically at 570 nm using a microplate reader and expressed as nmol/min/mL.
           
    For MDA measurement, 10 mg of liver tissue was homogenized in 300 µL of MDA Lysis Buffer supplemented with 3 µL of BHT. The homogenate was processed according to the manufacturer’s protocol and MDA levels were determined colorimetrically at 532 nm based on the formation of an MDA–thiobarbituric acid (TBA) adduct. Results were expressed as nmol/mg tissue.
           
    For ACOX1 measurement, 10 mg of liver tissue was homogenized in 100 µL of phosphate-buffered saline (PBS). The homogenate was processed according to the manufacturer’s protocol and the resulting supernatant was analyzed using the quantitative sandwich ELISA. Absorbance was measured at 450 nm using a microplate reader and ACOX1 concentrations were determined from the corresponding standard curve and expressed as ng/mL of liver-homogenate supernatant.
           
    For p53 measurement, 500 mg of liver tissue was homogenized in 500 µL of PBS. The homogenate was processed according to the manufacturer’s protocol and the resulting supernatant was analyzed using the sandwich ELISA. Absorbance was measured at 450 nm using a microplate reader and p53 concentrations were determined from the corresponding standard curve and expressed as ng/mL of liver-homogenate supernatant.
           
    For 8-OHdG measurement, serum samples were diluted 1:20 (v/v) in the supplied sample diluent and analyzed using the competitive ELISA according to the manufacturer’s protocol. Absorbance was measured at 450 nm using a microplate reader and 8-OHdG concentrations were determined from the standard curve, corrected for the dilution factor and expressed as pg/mL.
           
    Total protein concentration was not determined; therefore, CAT, ACOX1 and p53 values were reported in the assay-specific units generated according to the respective kit procedures rather than normalized to total protein. MDA levels were expressed as nmol/mg tissue based on the original tissue mass used for the assay. 
     
    Liver and kidney function tests
     
    Serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) activities and urea and creatinine concentrations were measured using an AU680 clinical chemistry analyzer (Beckman Coulter, Inc., Brea, CA, USA) with the manufacturer’s reagents and according to the manufacturer’s protocols.
     
    Statistical analysis
     
    Statistical analyses were performed using IBM SPSS Statistics, version 29.0 (IBM Corp., Armonk, NY, USA). Data are presented as mean±standard error of the mean (SEM). Differences among the six experimental groups were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s honestly significant difference (HSD) post hoc test for multiple comparisons. The assumptions of normality and homogeneity of variances were assessed using the Shapiro-Wilk and Levene’s tests, respectively. Statistical significance was set at P < 0.05. ANOVA results are reported as F statistics with the corresponding degrees of freedom and exact P values where available. All 10 animals per group were included in the analysis of each outcome, with no exclusions. Numerical mean ± SEM values for the measured outcomes are provided in Table 1. In the graphical presentation of the results, groups not sharing a common superscript letter were considered significantly different according to Tukey’s HSD test.

    Table 1: Numerical summary of measured biochemical and protein endpoints across experimental groups.

    RESULTS AND DISCUSSION

    Oxidative stress, oxidative DNA damage and ACOX1 response
     
    Hepatic MDA levels
     
    Hepatic MDA levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 39.179, P<0.001).  MDA levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with no significant difference between the two BPA-treated groups. Spirulina platensis co-treatment significantly reduced MDA levels in both HD+S and LD+S groups to values that did not differ significantly from the control. The S. platensis-only group also did not differ significantly from the control (Fig 1; Table 1).

    Fig 1: Hepatic malondialdehyde (MDA) levels.


     
    Hepatic catalase activity
     
    Hepatic CAT activity differed significantly among experimental groups (one-way ANOVA: F(5,54) = 190.596, P<0.001). CAT activity was significantly increased in the BPA-LD group compared with the control (P<0.05), whereas the BPA-HD group did not differ significantly from the control. S. platensis co-treatment significantly reduced CAT activity in the LD+S group compared with BPA-LD, although it remained significantly higher than the control. CAT activity in the HD+S and S. platensis-only groups did not differ significantly from the control (Fig 2; Table 1).

    Fig 2: Hepatic catalase (CAT) activity.



    Serum 8-OHdG levels
     
    Serum 8-OHdG, assessed as a circulating marker of oxidative DNA damage, differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 61.893, P<0.001). Serum 8-OHdG levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with significantly higher levels in BPA-LD than BPA-HD. S. platensis co-treatment significantly reduced 8-OHdG levels in both HD+S and LD+S groups compared with their respective BPA-only groups; however, both co-treatment groups remained significantly higher than the control. The S. platensis-only group did not differ significantly from the control (Fig 3; Table 1).

    Fig 3: Serum 8-hydroxy-2¢-deoxyguanosine (8-OHdG) levels.


     
    Hepatic ACOX1 levels
     
    Hepatic ACOX1 levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 224.280, P<0.001). ACOX1 levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with no significant difference between the two BPA-treated groups. S. platensis co-treatment significantly reduced ACOX1 levels in both HD+S and LD+S groups compared with their respective BPA-only groups. ACOX1 levels in HD+S did not differ significantly from the control, whereas LD+S remained significantly higher. The S. platensis-only group did not differ significantly from the control (Fig 4; Table 1).

    Fig 4: Hepatic acyl-CoA oxidase 1 (ACOX1) levels.


     
    Hepatic p53 protein levels
     
    Hepatic p53 protein levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 195.916, P<0.001). p53 protein levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with no significant difference between the two BPA-treated groups. S. platensis co-treatment significantly reduced p53 levels in both HD+S and LD+S groups compared with their respective BPA-only groups. Both co-treatment groups showed significantly lower p53 levels than the control and differed significantly from each other. The S. platensis-only group did not differ significantly from the control (Fig 5; Table 1).

    Fig 5: Hepatic p53 protein levels.


     
    Liver and kidney function markers
     
    Serum urea levels
     
    Serum urea levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 38.979, P<0.001). Urea levels were significantly increased in both BPA-HD and BPA-LD groups compared with the control (P<0.05), with significantly higher levels in BPA-LD than BPA-HD. S. platensis co-treatment was associated with lower mean urea levels in both co-treatment groups. Urea levels in HD+S did not differ significantly from BPA-HD but remained significantly higher than the control, whereas LD+S was significantly lower than BPA-LD and did not differ significantly from either the control or BPA-HD. The S. platensis- only group did not differ significantly from the control (Fig 6; Table 1).

    Fig 6: Serum urea levels.


     
    Serum creatinine levels
     
    Serum creatinine levels differed significantly among the experimental groups (one-way ANOVA: F(5,54) = 8.745, P = 0.001). Creatinine was significantly increased in the BPA-LD group compared with the control (P<0.05), whereas BPA-HD did not differ significantly from the control. S. platensis co-treatment significantly reduced creatinine in the LD+S group compared with BPA-LD, to a level that did not differ significantly from the control. Creatinine levels in the HD+S and S. platensis-only groups also did not differ significantly from the control (Fig 7; Table 1).

    Fig 7: Serum creatinine levels.


     
    Serum ALT and AST activities
     
    Serum ALT activity differed significantly among experimental groups (one-way ANOVA: F(5,54) = 112.523, P<0.001). ALT activity was significantly increased in the BPA-LD group compared with the control and all other experimental groups (P < 0.05), whereas BPA-HD, HD+S, LD+S and S. platensis-only groups did not differ significantly from the control. S. platensis co-treatment significantly reduced ALT activity in the LD+S group compared with the corresponding BPA-LD group (Fig 8; Table 1).

    Fig 8: Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities.


           
    Serum AST activity differed significantly among experimental groups (one-way ANOVA: F(5,54) = 27.186, P<0.001).  AST activity was significantly increased in the BPA-HD, BPA-LD, HD+S and LD+S groups compared with the control (P<0.05), with BPA-LD showing significantly higher activity than all other experimental groups. S. platensis co-treatment significantly reduced AST activity in the LD+S group compared with BPA-LD, whereas HD+S did not differ significantly from BPA-HD. The S. platensis-only group did not differ significantly from the control (Fig 8; Table 1).
           
    The present study shows that repeated oral exposure to BPA induces multiple biochemical alterations involving oxidative stress, circulating 8-OHdG, hepatic ACOX1 and p53 protein levels and markers of hepatic and renal function in male rats. These effects were accompanied by increased hepatic MDA and ACOX1 levels, differential changes in CAT activity, elevated circulating 8-OHdG and changes in serum ALT, AST, urea and creatinine. Co-administration of Spirulina platensis attenuated several of these responses, although  the degree of modulation varied according to the measured endpoint and BPA dose. Collectively, these findings support oxidative stress and altered circulating 8-OHdG as prominent biochemical responses to BPA exposure and indicate that S. platensis can modulate several biochemical responses associated with BPA exposure.
           
    One of the clearest effects of BPA exposure was the increase in hepatic MDA levels at both doses. MDA is widely used as an indicator of lipid peroxidation and reflects oxidative injury to cellular membranes. The elevation observed in the present study is consistent with previous experimental evidence showing that BPA can disturb cellular redox homeostasis and promote oxidative stress in different tissues (Manzoor et al., 2022; Rezg et al., 2014; Abdulhameed et al., 2022; Boudali et al., 2026; Ravikumar et al., 2022). Increased lipid peroxidation may result from excessive generation of reactive oxygen species together with an insufficient antioxidant response, ultimately affecting membrane integrity and cellular function. Importantly, co-treatment with S. platensis reduced MDA to levels that were not significantly different from the control, indicating attenuation of the BPA-associated increase in lipid peroxidation.
           
    The CAT response was more complex. CAT activity was markedly increased in the BPA-LD group, whereas the BPA-HD group did not differ significantly from the control. This response indicates that antioxidant enzyme activity did not increase proportionally with BPA dose. Increased CAT activity at the lower BPA dose may represent an adaptive response to enhanced hydrogen peroxide production, whereas the absence of a comparable increase at the higher dose may reflect a different balance between oxidative challenge and antioxidant capacity. Nevertheless, the present data do not establish the mechanism responsible for this pattern and this interpretation should therefore remain cautious.
           
    The increase in serum 8-OHdG is consistent with enhanced oxidative DNA damage; however, as a circulating biomarker, it does not identify the tissue source of DNA oxidation. Both BPA doses significantly increased 8-OHdG, with a greater response in the BPA-LD group than in the BPA-HD group. 8-OHdG is generated following oxidative modification of guanine residues and is commonly used as a marker of oxidative DNA damage. The parallel elevation of hepatic MDA and circulating 8-OHdG therefore suggests that BPA exposure was associated with both lipid peroxidation and systemic oxidative DNA damage. S. platensis significantly reduced 8-OHdG in both co-treatment groups; however, levels remained significantly higher than those of the control. Thus, unlike the near normalization observed for MDA, the modulatory effect of S. platensis on circulating 8-OHdG was only partial.
           
    Hepatic ACOX1 levels were also increased following exposure to both BPA doses. ACOX1 catalyzes the first and rate-limiting step of peroxisomal fatty-acid β-oxidation, a process that generates hydrogen peroxide as a metabolic by-product. Increased ACOX1 may therefore contribute to the altered hepatic oxidative environment observed following BPA exposure (Quijano et al., 2016). Co-administration of S. platensis markedly attenuated this response. ACOX1 in the HD+S group was comparable to the control, whereas the LD+S group remained significantly elevated. The differential responses of the two co-treatment groups further illustrate the endpoint-specific nature of the effects observed.
           
    A particularly relevant finding was the increase in hepatic p53 protein levels following BPA exposure. p53 is a central cellular stress-response protein that participates in the regulation of DNA-damage responses, cell-cycle control, DNA repair and apoptosis (Liu et al., 2020). The simultaneous increases in circulating 8-OHdG and hepatic p53 protein abundance observed in the BPA-treated groups are therefore consistent with cellular stress responses associated with oxidative condition.
           
    Co-administration of S. platensis significantly reduced hepatic p53 levels in both BPA-treated groups. Interestingly, p53 levels in the co-treatment groups were lower than those in the control group rather than merely returning to control values. Therefore, this response should not be interpreted as simple normalization of p53. Instead, it indicates substantial modulation of the hepatic p53 response during combined BPA and S. platensis exposure. Because downstream components of the p53 pathway were not assessed, the biological significance of this reduction cannot be established from the present data alone.
           
    The modulatory responses observed with S. platensis are consistent with its reported antioxidant properties. S. platensis contains several bioactive constituents, including phycocyanin, carotenoids, phenolic compounds, vitamins and other antioxidant components that may contribute to its ability to limit oxidative injury (Seyidoglu et al., 2020; Abdel-Daim et al., 2018; Finamore et al., 2017; Chouala et al., 2024). Previous experimental studies have similarly reported protective effects of S. platensis against chemically induced oxidative stress and tissue injury (Abdel-Daim et al., 2018; Finamore et al., 2017). Of particular relevance, Chouala et al., (2024) reported protection by Arthrospira platensis against BPA-induced nephrotoxicity in rats, providing relevant support for its potential to modulate BPA-associated toxicity.
           
    Changes in serum biochemical markers further indicated that BPA exposure was associated with biochemical alterations in organ-function markers beyond the oxidative stress endpoints. The BPA-LD group showed particularly pronounced elevations in ALT and AST, whereas the BPA-HD group exhibited a significant increase in AST without a corresponding significant increase in ALT. Similar associations between chemically induced hepatic oxidative stress, lipid peroxidation and alterations in serum liver enzymes have been reported in experimental rat models (Ayaz et al., 2017). Similarly, serum urea increased following both BPA doses, while creatinine was significantly elevated only in the BPA-LD group. Co-administration of S. platensis attenuated several of these alterations, particularly in the BPA-LD group, although the extent of attenuation varied among biomarkers.
           
    The apparently stronger responses to the lower BPA dose for several endpoints, including CAT, 8-OHdG, ALT, AST, urea and creatinine, warrant careful interpretation. BPA has been reported to produce non-monotonic dose-response patterns under some experimental conditions (Vandenberg et al., 2012; Vandenberg, 2014; Welshons et al., 2003). However, the present experiment was not specifically designed to characterize the shape or mechanism of a non-monotonic dose-response relationship. Accordingly, the observed differences between the two BPA doses should be regarded as endpoint-specific responses rather than definitive evidence of a particular dose-response mechanism.
           
    It is also important to place the doses used in the present study in the appropriate toxicological context. The BPA doses of 120 and 240 mg/kg/day are substantially higher than typical human environmental exposures and were selected based on previous toxicological work using these dose levels (Samova et al., 2018). Consequently, the findings should not be directly extrapolated to expected effects at ordinary human exposure levels. Rather, the study provides experimental evidence regarding biological responses to short-term, high-dose BPA exposure and the extent to which S. platensis modifies those responses.
           
    Several limitations should be considered. First, only male rats were examined, limiting extrapolation to females or other biological contexts. Second, the 21-day exposure period represents a relatively short toxicological model and does not address chronic exposure. Third, the BPA doses were supra-environmental and therefore do not reproduce typical human exposure conditions. Fourth, the mechanistic assessment was limited to selected markers of oxidative stress, peroxisomal metabolism and p53 protein abundance. Histopathological evaluation, direct tissue-based assays and additional molecular markers of DNA repair, apoptosis, inflammation and antioxidant signaling would provide a more comprehensive characterization of tissue response. In addition, serum 8-OHdG is a systemic biomarker and does not localize DNA oxidation to hepatic tissue, while increased p53 protein abundance alone does not establish p53-pathway activation or apoptosis. Finally, the study demonstrates biochemical associations but cannot establish direct causal relationships among ACOX1 levels, oxidative stress, DNA damage and p53 signaling.

    CONCLUSION

    Repeated oral exposure to BPA induced oxidative stress, elevated serum 8-OHdG consistent with oxidative DNA damage, altered hepatic p53 protein levels and changes in liver and kidney function markers in male rats. Co-administration of Spirulina platensis attenuated several BPA-associated biochemical changes, particularly hepatic lipid peroxidation and serum 8-OHdG, increased hepatic ACOX1 levels, the BPA-associated increase in hepatic p53 protein levels and selected biochemical indices of hepatic and renal function. However, the magnitude of modulation varied among endpoints and several responses did not increase proportionally with BPA dose. These findings indicate that S. platensis modulates multiple biochemical responses associated with BPA exposure in this experimental model, while highlighting the need for further studies using environmentally relevant BPA doses, longer exposure periods, histopathological assessment and broader molecular analyses to clarify the mechanisms underlying these modulatory effects.

    ACKNOWLEDGEMENT

    The authors extend their appreciation to the Ongoing Research Funding Program (ORF-2026-811), King Saud University, Riyadh, Saudi Arabia, for funding this work. The authors also thank the staff of the Animal House Facility, Department of Zoology, King Saud University, for their technical support and assistance with animal care.
     
    Funding
     
    This work was supported by the Ongoing Research Funding Program (ORF-2026-811), King Saud University, Riyadh, Saudi Arabia.
     
    Animal ethics
     
    The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at King Saud University, Riyadh, Saudi Arabia (Approval No. KSU-SE-25-38). All experimental procedures were conducted in accordance with the approved institutional guidelines for the care and use of laboratory animals.
     
    Author contributions
     
    Lama A. Al-Otaibi and Muath Q. Al-Ghadi conceived the study. Lama A. Al-Otaibi and Promy Virk contributed to the methodology. Lama A. Al-Otaibi performed the experiments, conducted the laboratory procedures and collected and curated the data. Promy Virk contributed to validation and study supervision. Muath Q. Al-Ghadi supervised the research and administered the project. Lama A. Al-Otaibi prepared the original draft of the manuscript. Promy Virk and Muath Q. Al-Ghadi reviewed and revised the manuscript for important intellectual content. All authors read and approved the final version of the manuscript.
     
    Data availability statement
     
    The data supporting the findings of this study are available from the corresponding author upon reasonable request.

    CONFLICT OF INTEREST

    On behalf of all co-authors, we confirm that the authors declare no conflicts of interest, financial or otherwise, that could have influenced the research presented in the manuscript.

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