Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used worldwide. These substances are commonly prescribed as analgesics in human medicine and are also frequently available without a prescription as over-the-counter medications. Because of their extensive use, NSAIDs have become a major class of environmental contaminants. Their widespread environmental occurrence has raised concern about potential risks to human health [1]. Accordingly, NSAIDs are classified as emerging contaminants. These compounds are usually detected in the environment at parts-per-trillion levels (ng/L or ng/kg), far below therapeutic doses and typical exposure levels [2].

Naproxen sodium, an NSAID derived from propionic acid, is primarily prescribed for osteoarthritis and rheumatoid arthritis and is also used as an antipyretic and analgesic agent. It is among the most frequently detected pharmaceuticals in environmental matrices, including aquatic systems and agroecosystems, along with other compounds such as carbamazepine, sulfamethoxazole, ibuprofen, gemfibrozil, atenolol, diclofenac, erythromycin, and bezafibrate. Because of incomplete degradation, naproxen occurs in groundwater, surface water, and drinking water, posing a potential threat to aquatic organisms through chronic exposure and to humans who consume these organisms [3]. Naproxen is poorly metabolized by humans, and wastewater treatment plants lack microbial strains capable of degrading it efficiently. In addition, naproxen sodium readily undergoes photolysis, and its phototransformation products are often more toxic than the parent compound. As a result, both naproxen and its transformation products are released into the environment [4]. In aquatic organisms, exposure to naproxen at environmental concentrations has been associated with altered enzymatic activity and gene expression, histopathological changes, cellular damage, and reproductive abnormalities. Naproxen can also affect mRNA expression and cause gastrointestinal and renal effects in zebrafish. Beyond aquatic ecosystems, naproxen may pose risks to human health, including DNA damage, congenital abnormalities, gastric ulcers, chronic depressive disorders, respiratory impairment, and cardiovascular and renal diseases [5].

The combined presence of pharmaceuticals in the environment highlights the need to assess their interactive effects. Cleuvers [6] demonstrated that predicting the toxicity of pharmaceutical mixtures is critical because these substances rarely occur in isolation. Studies have shown that naproxen interacts with other drugs, such as carbamazepine and sulfamethoxazole, and may affect amphibian growth and development at environmental concentrations [7].

Given the limited toxicological evidence on the ovarian toxicity of naproxen sodium at environmentally relevant doses, this study aimed to elucidate how long-term exposure to this chemical, individually or in a realistic mixture, may disrupt ovarian function and integrity in female Wistar rats.

Environmental pollution driven by industrial and pharmaceutical activities is a growing global concern. Compounds such as NSAIDs, pesticides, and personal care products are major contributors to environmental contamination. However, current chemical risk assessment strategies often fail to capture the complexity of real-life human exposure to mixtures of these substances [18]. Despite their widespread environmental presence, the potential adverse effects of NSAIDs, alone or in combination with other pollutants, on reproductive health at environmentally relevant concentrations remain underexplored. This gap underscores the need for toxicological studies that simulate real-world environmental exposure. Therefore, the present study assessed the effects of long-term exposure to naproxen alone or in a mixture with diclofenac, ibuprofen, and trifloxystrobin administered in drinking water at environmental concentrations on ovarian health, based on BWG, ovary index, progesterone, MDA, CAT, IL-6, Bcl-2 expression, and primordial and preantral follicle counts. To approximate environmentally relevant oral exposure, female rats were exposed to these contaminants through drinking water.

The observed increase in the ovary index after long-term exposure in both treated groups may represent an early biomarker of reproductive toxicity. Previous research has demonstrated that NSAIDs may impair normal ovarian function by disrupting prostaglandin synthesis, leading to tissue changes such as edema, inflammation, or ovarian hypertrophy [19]. This increase in ovary index may be attributable to oxidative stress or inflammation-driven mechanisms that promote tissue remodeling and structural alterations.

Oxidative stress is defined as an imbalance between pro-oxidant and antioxidant species that causes molecular and cellular damage [20]. In the present study, two oxidative stress biomarkers were assessed: MDA and CAT. Treatment had no significant effect on MDA levels, although previous studies of subchronic and chronic NSAID exposure have reported significantly decreased MDA levels [21,22].

Regarding CAT activity, a key first-line antioxidant defense against reactive oxygen species [23], our results showed a marked reduction in both treated groups, suggesting impaired antioxidant capacity and redox imbalance, consistent with previous research [24]. Such redox disturbances may contribute to cellular damage and interfere with endocrine signaling pathways, thereby potentially impairing reproductive hormone synthesis and regulation. In female reproduction, these hormones regulate folliculogenesis, oogenesis, estrous cycling, sexual behavior, and ovulation [25]. Our results demonstrated a significant decrease in serum progesterone levels in the treated groups. Consistent with these findings, NSAIDs have been shown to cause luteinized unruptured follicle syndrome, preventing corpus luteum formation and subsequent progesterone production [26,27]. Moreover, pesticides have been documented to act as endocrine disruptors and induce oxidative stress-mediated hormonal dysregulation [28].

IL-6 is a key proinflammatory cytokine that is often upregulated during tissue injury and oxidative stress. It has been implicated in the regulation of ovarian function, including follicular development, ovulation, and luteal maintenance [29,30]. In the present study, IL-6 levels did not differ significantly among groups, suggesting that chronic exposure to naproxen under the present experimental conditions did not elicit a pronounced inflammatory cytokine response. However, IL-6 expression is tightly regulated and may not increase unless a substantial inflammatory or immune trigger is present.

Female fertility depends on ovarian follicles, which have two main functions: generating mature oocytes and synthesizing steroid hormones [31]. Follicular pool development and follicular growth are essential for fertility and full reproductive function. In humans, follicles regularly leave dormancy and begin a developmental process that takes approximately 1 year. This growth occurs in two phases: a gonadotropin-independent stage (from primordial to secondary follicles) that occurs throughout life, and a gonadotropin-dependent stage (from antral to ovulatory follicles) that begins after puberty [32,33]. In the histological analysis, we observed a significant reduction in healthy preantral follicle numbers in both Nap without a significant effect on the primordial reserve. This decline indicates that chronic exposure to naproxen may impair normal folliculogenesis, potentially by interfering with ovarian follicle recruitment, growth, and maturation. These findings are consistent with earlier studies demonstrating that NSAIDs and other endocrine-disrupting chemicals can adversely affect ovarian function by inhibiting prostaglandin synthesis, which is essential for follicular development and ovulation [34,35]. These disturbances can provoke short-term menstrual or estrous-cycle irregularities and, over time, may deplete the ovarian reserve and reduce fertility [36]. Observational studies of women undergoing assisted reproduction are broadly consistent with these findings, linking higher body burdens of common endocrine-disrupting chemicals, including bisphenol A, phthalates, and per- and polyfluoroalkyl substances, to diminished ovarian reserve and poorer in vitro fertilization outcomes [37].

Bcl-2 is an anti-apoptotic protein in the Bcl-2 family, a group of essential regulators of the intrinsic, or mitochondrial, apoptotic pathway. It suppresses programmed cell death largely by preserving mitochondrial membrane integrity and inhibiting cytochrome c release, thereby favoring cell survival. The balance between anti-apoptotic proteins such as Bcl-2 and pro-apoptotic members of the same family plays a vital role in determining cell fate under normal and pathological conditions [38]. In the present study, Bcl-2 expression was evaluated in both antral follicles and ovarian stromal compartments. Bcl-2 immunoreactivity was significantly reduced in the treated groups, with a more pronounced decrease in the mixture-exposed group across both compartments, suggesting greater pro-apoptotic susceptibility associated with combined chemical exposure. Given the central role of Bcl-2 in granulosa cell survival, its downregulation indicates increased vulnerability of ovarian cells to apoptotic signaling, which may compromise follicular development and ovarian functional integrity. These observations are consistent with the histomorphometric findings of reduced follicle populations and increased follicular atresia [39]. Similar patterns have been reported after exposure to methoxychlor, in which suppression of Bcl-2 expression was associated with increased follicular atresia and apoptotic signaling [40]. In addition, alterations in stromal cell survival may further exacerbate follicular loss by disrupting the ovarian microenvironment required for follicular support. The effects of naproxen—alone or in combination—on cell survival may be mediated, at least in part, by cyclooxygenase inhibition. This may result in reduced prostaglandin synthesis and downstream modulation of anti-apoptotic signaling, including Bcl-2 expression.

Collectively, these data indicate that long-term exposure to environmentally relevant doses of naproxen, alone or in a mixture, disrupted ovarian homeostasis without altering systemic inflammation, lipid peroxidation, or body weight. Treated groups exhibited impaired antioxidant defenses, as indicated by reduced CAT activity, diminished progesterone levels, and marked declines in growing follicles and anti-apoptotic Bcl-2 expression. Notably, these adverse effects on follicular development and cellular survival were most pronounced in the mixture group, suggesting that combined exposures may exert additive or interactive effects that warrant further mechanistic investigation.

This study has several limitations that should be considered. Although progesterone was measured as an indicator of ovarian endocrine activity, other reproductive hormones, such as estradiol, follicle-stimulating hormone, luteinizing hormone, and anti-Müllerian hormone, were not evaluated. In the present study, progesterone was selected as the primary marker of luteal function and ovarian activity after chronic exposure. However, including additional hormonal parameters would provide a more comprehensive characterization of endocrine regulation during folliculogenesis. Furthermore, apoptosis was assessed by immunohistochemical analysis of Bcl-2 expression; however, complementary assays, such as caspase-3 activity or terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining, were not performed. Although these analyses were beyond the scope of the present study, they should be considered in future investigations to further elucidate the molecular mechanisms involved in ovarian toxicity.