Combined Effects of 7,12-dimethylbenz[a]anthracene (DMBA) with Cobalt, Cadmium, or Zinc on Fertility in Rats.

Infertility is defined as the failure to achieve pregnancy after 12 months of regular, unprotected sexual intercourse ( 1 ). Approximately 85% of couples who are unable to conceive have an identifiable cause of infertility, with ovulatory disorders, male-related factors, and tubal abnormalities being the primary contributors. The remaining 15% are classified as having "unexplained infertility" ( 2 ). The prevalence of infertility is increasing worldwide, prompting growing attention to environmental factors, as genetic determinants alone are insufficient to fully explain the phenomenon. Female infertility is multifactorial and may result from genetic mutations, chromosomal abnormalities, lifestyle factors, ovulatory dysfunction, tubal pathologies, endometriosis, or unexplained causes. Recent studies highlight the influence of lifestyle on reproductive health, indicating that unhealthy behaviors, including poor dietary habits, stress, alcohol consumption, smoking, and obesity, may negatively affect female physiology and reduce the likelihood of conception ( 3 , 4 ). Environmental exposures have also been extensively investigated for their impact on fertility, 7,12-Dimethylbenz[a]anthracene (DMBA) is commonly used as a model carcinogen to induce mammary tumors in rodents because it mimics the genotoxic and endocrine-disrupting effects of numerous environmental pollutants. Although direct human exposure to DMBA is rare, this compound serves as a standardized experimental tool to investigate how oxidative DNA damage and BRCA1 dysregulation contribute to reproductive toxicity. Its use therefore provides mechanistic insight rather than representing a real-world exposure scenario, enabling controlled assessment of the combined effects of environmental stressors on fertility ( 5 , 6 ). In contrast, heavy metal exposure remains a significant and ongoing concern for the general population. Cadmium (Cd), cobalt (Cd), and zinc (Zn) are trace elements found in food, water, and industrial emissions, and their accumulation has been documented across various age groups, particularly among individuals residing near industrial areas, consuming contaminated food, or employed in metal-related occupations ( 7 , 8 ). Epidemiological studies indicate that low-level, long-term exposure is now widespread, particularly among women of reproductive age, raising concerns about cumulative impacts on ovarian function and early pregnancy outcomes. While Zn has been associated with enhanced fertility due to its antioxidant properties, Cd and Co are established reproductive toxicants ( 9 , 10 ). Therefore, investigating their interaction with a genotoxic agent such as DMBA provides new insights into how environmental co-exposures may synergistically reproductive capacity via oxidative stress and apoptosis. Oxidative stress occurs when the production of free radicals and reactive oxygen species (ROS) exceeds the capacity of the body’s antioxidant defenses ( 11 ). An increase in ROS and/or a decrease in antioxidant levels leads to oxidative stress, resulting in damage to cellular components, apoptosis, and necrosis ( 12 ). Lipid peroxidation is a key indicator of oxidative damage and can be quantified by measuring malondialdehyde (MDA), the end product of lipid peroxidation. The overproduction of ROS can be triggered by multiple factors, which are generally classified as external or internal ( 13 ). Notably, exposure to environmental toxins, including heavy metals and organic compounds, is a well-established external cause of oxidative stress in biological systems ( 14 ). Antioxidants, which counteract ROS, originate from both endogenous and dietary sources. Endogenous antioxidants include enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), as well as small molecules like glutathione (GSH), bilirubin, and uric acid ( 15 , 16 ). The combined activity of these antioxidants contributes to overall protection against oxidative damage and helps maintain cellular homeostasis ( 13 , 17 ). The breast cancer gene 1 ( Brca1 ) mutation accounts for 40-45% of hereditary breast cancer cases worldwide ( 18 ). Brca1 encodes a phosphoprotein essential for maintaining genomic integrity through DNA repair, chromatin remodeling, and cell-cycle regulation, thereby functioning as a critical tumor suppressor ( 19 ). Beyond its established role in carcinogenesis, Brca1 also plays a pivotal part in reproductive physiology. It is expressed in oocytes and granulosa cells, where it safeguards the ovarian reserve by repairing DNA double-strand breaks during folliculogenesis. Deficiency or mutation in Brca1 accelerates oocyte attrition, induces premature ovarian failure, and reduced fertility, as demonstrated in both human and animal studies. Consequently, Brca1 represents a key molecular link between mammary tissue toxicity and reproductive dysfunction, making it an ideal biomarker for exploring how environmental genotoxic stressors, such as DMBA and heavy metals, can concurrently impair fertility and mammary gland health ( 20 ). While DMBA and heavy metals such as Cd, Co, and Zn are individually known to induce cellular toxicity and disrupt endocrine functions, their combined effects remain poorly understood. Investigating these interactions is crucial, as simultaneous environmental exposures may produce synergistic or antagonistic effects on fertility and mammary tissue integrity. This study was therefore designed to evaluate the environmental impact on fertility in rats by examining the combined effect of DMBA with Cd, Co, and Zn on systemic redox balance and Brca1 expression.

Fertility outcomes were assessed in terms of pregnancy rates, defined as the percentage of mated females that became pregnant: The pregnancy rates for the Control, DMBA, Cd-, Co-, and Zn-treated groups were 100%, 20%, 30%, 60%, and 60%, respectively. For fertility assessment, each female rat was paired with a proven fertile male at a 1:1 ratio for a continuous 10-day mating period following the treatment phase. Vaginal smears were examined daily for the presence of sperm to confirm successful mating (considered day 0 of pregnancy). Each female had multiple mating opportunities during this observation period. Blood sampling and pregnancy outcomes were recorded for all females that mated successfully. The duration of pregnancy was shorter in DMBA- (17 days), Cd- (18 days), Co- (19 days), and Zn-treated (20 days) rats compared to the control rats (21 days) (P=0.018). The number of pups born per dam (litter size) was 21 in the control group, 6 in the Cd-treated group, 7 in the Co-treated group, and 10 in the Zn-treated group. The sex ratio, calculated as (number of male pups/total number of pups)×100, was 88.88%, 40.00%, 33.33%, 44.44%, and 44.44% for the control, DMBA-, Cd-, Co-, and Zn-treated groups, respectively. The levels of oxidative stress indicators, including MDA, SOD, GSH, and CAT, are presented in Table 1 and Figure 1 as mean ± standard deviation (SD). The MDA level was significantly elevated (P<0.001) in the DMBA-treated group, the Cd/DMBA-treatedgroup, and the Co/DMBA-treated group compared with the control group. In contrast, the Zn/DMBA-treated group showed no significant difference from the control (P<0.001). Furthermore, MDA levels were significantly higher in the Cd/DMBA- and Co/DMBA-treated groups compared to the DMBA-treated group, whereas the Zn/ DMBA-treated group showed no significant difference from DMBA alone. The SOD level was significantly decreased (P<0.001) in the DMBA-treated, Cd/DMBA-treated, Co/DMBA -treated, and Zn/DMBA-treated groups compared with the control group. Moreover, SOD levels were significantly lower in the Cd/DMBA- and Co/DMBA -treated groups compared with the DMBA-treated group, whereas the Zn/ DMBA-treated group exhibited significantly higher SOD levels compared with the DMBA-treated group. The GSH level was significantly decreased (P<0.001) in the DMBA-treated, Cd/DMBA-, Zn/DMBA, and Co/ DMBA-treated groups compared with the control group. Furthermore, GSH levels were significantly lower in the Cd/DMBA-, Co /DMBA-, and Zn /DMBA-treated groups compared with the DMBA-treated group. Oxidative stress markers in rat groups Values are expressed as mean ± SD. MDA; Malondialdehyde, SOD; Superoxide dismutase, GSH; Glutathione, CAT; Catalase, DMBA; 7,12-Dimethylbenz[a]anthracene, Cd; Cadmium, Zn; Zinc, Co; Cobalt, *; Significant compared with control (P≤0.05), and #; Significant compared with DMBA (P≤0.05). Statistical analysis was performed using one-way ANOVA followed by LSD post hoc test. Representative immunohistochemistry images of mammary gland tissues showing BRCA1 expression. Red arrows indicate BRCA1-positive nuclei (dark brown staining). A, B. Control group replicates, C, D. DMBA-treated group replicates, E, F. Cd/DMBA-treated group replicates, G, H. Zn/DMBA-treated group replicates, I, J. Co /DMBA-treated group replicates (magnification 40×, scale bar: 50 µm). DMBA; 7,12-Dimethylbenz[a]anthracene, Cd; Cadmium, Zn; Zinc, and Co; Cobalt. The CAT level was significantly decreased (P<0.001) in the DMBA-treated, Cd/DMBA-treated, Co/DMBA -treated, and Zn/DMBA-treated groups compared with the control group. Furthermore, CAT levels were significantly lower in the Cd/DMBA- and Co/DMBA-treated groups compared with the DMBA-treated group, whereas the decrease in CAT in the Zn/DMBA-treated compared with the DMBA alone was not significantly. The total number of cells was significantly reduced in the DMBA-, Cd/DMBA-, Zn/DMBA-, and Co/DMBA -treated groups compared to the control group. Moreover, all trace element/DMBA combination groups showed a significant reduction in total cell number compared with the DMBA-treated group. The Co/DMBA-treated group exhibited the highest levels of apoptosis and necrosis, which were significantly higher (P<0.001) compared with both the control and DMBA-only groups ( Table 2 ). In addition, the Cd/ DMBA- and Zn/DMBA-treated groups showed a significant increase in the number of apoptotic and necrotic cells relative to the control group. TUNEL assay revealed marked differences in DNA fragmentation among the experimental groups. The control group exhibited minimal apoptotic activity, whereas exposure to DMBA induced a noticeable increase in TUNEL-positive nuclei. The Cd/DMBA group exhibited the highest level of DNA fragmentation, indicating pronounced cytotoxicity. In contrast, Zn/DMBA treatment markedly reduced the number of apoptotic cells compared with the DMBA-only group, suggesting a protective effect of zinc. The Co / DMBA group demonstrated an intermediate apoptotic response, consistent with a moderate modulatory effect of cobalt ( Fig .2 ). Nuclei stained positive (highlighted with yellow) by the TUNEL assay indicate DNA fragmentation (cell death). A, B. Control group replicates, C, D. DMBA -treated, E, F. Cd+DMBA-treated, G, H. Zn+DMBA-treated, I, J. Co+DMBA-treated groups. Arrows indicate representative apoptotic nuclei (scale bar: 50 µm). Apoptotic and necrotic cell counts in mammary gland tissues of rats Data are presented as mean ± SD. Means were compared using one-way ANOVA followed by LSD test. DMBA; 7,12-Dimethylbenz[a]anthracene, Cd; Cadmium, Zn; Zinc, Co; Cobalt, *; Significant vs. control, and #; Significant vs. DMBA. Cell counts were performed in five randomly selected fields per well at 400× magnification (~0.035 mm² per field). Breast cancer cell viability was significantly affected by the treatments ( Table 3 ). DMBA alone reduced cell viability compared with the control, while co-treatment with metal ions produced differential effects. Co enhanced the cytotoxicity of DMBA, whereas Zn partially mitigated its cytotoxic effects. Cd also exerted a moderate cytotoxic effect when combined with DMBA. These results indicate that the type of metal ion can modulate the cellular response to DMBA, highlighting the complex interplay between environmental factors and breast cancer cell viability ( Fig .3 ). Effect of treatments on breast cancer cell viability Data are presented as mean ± SD. The means were compared using one-way ANOVA followed by LSD test. DMBA; 7,12-Dimethylbenz[a]anthracene. Phase-contrast micrographs of MCF-7 cells after 24 hours treatment (200×). A. Control: confluent spindle-shaped monolayer, B. DMBA: cells showing partial rounding and intercellular gaps (arrowheads), C. Co/DMBA: moderate preservation of morphology with fewer gaps, D. Zn/DMBA: minimal morphological alteration, cell-cell contacts largely maintained, E. Cd/DMBA: pronounced cell rounding and detachment. Yellow arrowheads indicate representative cells exhibiting morphological changes, including rounding, detachment, or altered cell boundaries. DMBA; 7,12-Dimethylbenz[a]anthracene, Cd; Cadmium, Zn; Zinc, and Co; Cobalt.

This study investigated the impact of environmental toxicants, specifically DMBA and selected heavy metals (Cd, Co, Zn), on female fertility and mammary gland structure, focusing on mechanisms involving oxidative stress and gene expression. The primary objective was to explore how combined environmental exposures may simultaneously impair reproductive performance and increase susceptibility of mammary tissue to potential transformation. The etiological factors underlying infertility remain incompletely understood, with both genetic and environmental contributors playing key roles. DMBA is wellknown to induce genetic mutations in mammary tissue, leading to breast cancer ( 24 ). Evidence regarding infertility as a risk factor for breast cancer is conflicting ( 25 , 26 ), although breast cancer itself can lead to infertility, primarily due to chemotherapy ( 27 ). Additionally, BRCA1 mutations have been associated with occult premature ovarian failure ( 28 ). Previous studies have demonstrated that DMBA can induce ovarian toxicity in rodents, potentially via the Hippo signaling pathway and oxidative stress ( 29 ). DMBA exposure has also been shown to deplete oocytes in small follicles and elevate BAX protein levels in mouse ovarian primordial follicle oocytes postnatally ( 30 ). Collectively, these findings suggest that DMBA can compromise ovarian and mammary gland integrity through oxidative stress, thereby contributing to increased apoptosis and necrosis ( 31 ). Cd is known to exacerbate oxidative toxicity by indirectly promoting the overproduction of ROS ( 32 ). The impact of Cd on oxidative stress depends on exposure duration, with acute exposure generating free radicals such as superoxide anion, hydrogen peroxide, hydroxy radical, and lipid radicals. Several factors modulate cadmium-induced ROS production, including glutathione depletion, Kupffer cell activation, inflammatory conditions, and participation of metals in Fenton reactions ( 33 ). In mammary gland tissue, Cd has been reported to induce inflammation and apoptosis via the circ08409/miR -133a/TGFB2 axis ( 34 ). While a study documented cadmium-induced male infertility ( 35 ), evidence regarding its effects on female infertility remains limited. Notably, even trace concentrations of Cd and lead (Pb) have been implicated in female infertility ( 36 ). In our study, Cd coexposure with DMBA led to the highest levels of oxidative stress among the experimental groups, although the corresponding increases in apoptosis and necrosis were not the highest. These findings suggest that cadmium-induced oxidative stress does not always directly translate into proportional cell death, highlighting the complexity of its biological effects. Co is another element known to stimulate oxidative stress. In our study, DMBA/Co-treated rats exhibited a notable increase in oxidative stress compared to rats treated with DMBA alone. Correspondingly, apoptosis in the mammary gland was highest among all experimental groups, suggesting a strong cytotoxic effect of Co in combination with DMBA. Experimental studies have shown that Co can interfere with DNA repair mechanisms and directly induce DNA damage, DNA-protein crosslinking, and sister-chromatid exchange. Animal studies indicate that Co(II) compounds possess carcinogenic potential and can enhance UV-induced genetic alterations in V79 Chinese hamster cells ( 37 ). These toxic effects are likely mediated by cobalt’s capacity to generate hydroxyl radicals through Fentonlike reactions ( 38 ). Regarding reproductive outcomes, six out of ten female rats treated with DMBA/Co achieved pregnancy, indicating that Co co-exposure does not completely abolish fertility but may modulate reproductive performance. The combination of Zn and DMBA appeared to moderately improve redox balance. In our study, MDA levels in Zn/DMBA-treated rats were lower than in DMBA-only rats, approaching values similar to those of the control group. However, the antioxidants enzymes SOD and GSH remained significantly reduced, while CAT levels did not differ significantly from the DMBA group. Previous studies support these findings. Gulbahce-Mutlu et al. ( 39 ) reported that DMBA treatment significantly increased MDA levels in both plasma and mammary gland tissue, and co-treatment with Zn reduced MDA levels. They also observed a systemic and local increase in GSH levels with DMBA/Zn treatment. Despite the partial improvement in oxidative stress markers, apoptosis in the mammary glands of DMBA/Zn -treated rats was higher than in DMBA-treated rats. This suggests that oxidative stress alone may not fully account for the induction of apoptosis and necrosis, and other pathways may contribute to mammary gland cell death in this model. This study had several limitations. The sample size was relatively small, and there was no long-term follow-up, which may limit the interpretation of the results. Additionally, the use of a single cell line and a single animal species may restrict the generalizability of the findings to human. Further studies should include larger sample sizes, multiple animal models, and longer observation periods to validate and expand upon these findings.

Exposure to environmental toxicants, specifically trace elements such as zinc, cadmium, and cobalt, can negatively impact fertility in females with Brca1 mutations. In this study, DMBA was used to induce Brca1 -associated changes in female rats. Cd exerted a particularly strong effect on reproductive performance, inducing infertility in 7 of 10 rats at a low concentration (15 ppm). Oxidative stress was markedly elevated with Cd and Co exposure, whereas Zn showed a milder effect. Despite these differences, all three metals in combination with DMBA increased apoptosis and necrosis in mammary gland tissue. These findings indicate that trace element toxicity can compromise mammary gland integrity and reduce fertility in Brca1 -compromised females, highlighting the importance of environmental exposures in reproductive and breast health

This experimental study was conducted at the Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Science and Technology, University of Isfahan, Iran, from June 2021 to December 2023. All experimental procedures involving animals were reviewed and approved by the Research Ethics Committee of the University of Isfahan (IR.UI.REC.1403.121). The study was performed in accordance with the guidelines of the World Medical Association Declaration of Helsinki and the National Research Council’s Guide for the Care and Use of Laboratory Animals, ensuring the ethical treatment and welfare of all animals used in the experiment. DMBA (6 mg/mL; CAS No. 57-97-6, Sigma-Al-drich, USA) was prepared in deionized water. Cadmium nitrate (Cd (NO 3 ) 2 , Cat. No. 208185), cobalt chloride (CoCl 2 , Cat. No. C8661), and zinc chloride (ZnCl2, Cat. No. Z0152, Sigma-Aldrich, USA) were each dissolved in deionized water to prepare working solutions of 15 µg/mL Cd (NO 3 ) 2 , and 3 mg/mL CoCl 2 and ZnCl 2 . The study was approved by the Research Ethics Committee of the University of Isfahan (IR. UI.REC.1403.121) and was conducted in accordance with the World Medical Association Declaration of Helsinki for laboratory animal experiments. Fifty mature female Wister rats (150 ± 10 g) were acclimated for 2 days in well-ventilated polypropylene cages, with no more than five rats per cage. Rats were provided with a low-carbohydrate diet and water ad libitum. Ten male rats were included for mating purposes; all were pre-screened and confirmed fertile in a prior pilot study conducted under identical housing conditions to minimize variability due to male infertility. After acclimation, female rats were randomly assigned to five groups (n=10 per group): 1. Control: 0.05% sucrose (placebo) 2. DMBA only: 2 mL of 6 mg/mL DMBA (80 mg/kg) (57-97-6., Merck, Germany) 3. DMBA+Co: 2 mL of 6 mg/mL DMBA+2.5 mL of 3 mg/mL cobalt chloride (50 mg/kg) (7646-79-9., Merck, Germany) 4. DMBA+Zn: 2 mL of 6 mg/mL DMBA+2.5 mL of 3 mg/mL zinc chloride (50 mg/kg) (7646-85-7., Merck, Germany) 5. DMBA+Cd: 2 mL of 6 mg/mL DMBA+1 mL of 15 µg/mL cadmium nitrate (100 µg/kg) (10022-68-1., Merck, Germany) All substances were administered daily via oral gavage for 20 weeks. At the end of the study, rats were euthanized with ketamine (3 mL/kg) and xylazine (0.1 mL/kg). At the end of the 20-week experimental period, blood samples were drawn from each rat via the retro-orbital sinus under light anesthesia with ketamine (80 mg/kg) and xylazine (10 mg/kg). Approximately 2 mL of blood was collected from each animal using a sterile capillary tube and transferred into plain microtubes. The samples were centrifuged at 3,000 rpm for 10 minutes using a Hettich EBA 200 microcentrifuge (Hettich GmbH, Germany) to separate the serum. Serum levels of MDA, SOD, GSH, and CAT were measured using ELISA kits (Biont, China; Cat. Nos. E-EL-0060 for MDA, E-EL-0160 for SOD, E-EL-0023 for GSH, and E-EL-0042 for CAT) with an ELISA microplate reader (Human Diagnostics, Germany, Model: HumaReader HS). All serum samples were analyzed individually (not pooled), and each test was performed in triplicate (n=3 technical replicates per sample) to ensure accuracy and reproducibility. Mammary gland tissues were collected to assess BRCA1 protein expression. Tissues were fixed using Bouin’s solution (HT101128., Merck, Germany), dehydrated in graded ethanol (64-17-5., Merck, Germany), cleared in xylene (95-47-6., Merck, Germany)., and embedded in paraffin (8002-74-2., Merck, Germany). Sections of 5 µm thickness were cut from the tissue blocks, rehydrated, deparaffinized, and washed with phosphate-buffered saline (PBS, P3563., Merck, Germany). Endogenous peroxidase activity was blocked by incubating the sections in methanol (67-56-1., Merck, Germany) containing 1% hydrogen peroxide (7722-84-1, Merck, Germany) for 30 minutes. A PAP pen (Cat. No. GTX22601, Sigma, UK) was used to mark the area around each section to maintain consistent reaction conditions. Non-specific binding was prevented by incubating sections in PBS containing 1% bovine serum albumin (BSA, Cat No. A7906, Sigma, UK) for 45 minutes. Sections were then incubated at 4˚C with a rat polyclonal anti -BRCA1 antibody, clone I-20 (Cat. No. sc-135732, Santa Cruz Biotechnology, USA). Signal amplification was performed using the catalyzed signal amplification (CSA) kit (CSA Kit-DAKO Cat. No. K1500, DakoCytomation, UK) according to the manufacturer’s instructions. Finally, sections were counterstained with Harris hematoxylin (Cat. No. 109253, Sigma, USA) and rinsed thoroughly in running tap water for 5 minutes to remove excess dye. Slides were subsequently dehydrated through a graded series of alcohols (70%, 95%, and absolute ethanol), cleared in xylene, and mounted using Distyrene, Plasticizer, Xylene (DPX) mounting medium. Apoptosis and necrosis were assessed using a TUNEL enzyme assay kit (E-CK-A320., ELabScience, USA) following the manufacturer’s protocol. The human breast cancer cell line MCF-7 (Cat. No. D6434, Sigma, UK) was used in this study. Cells were cul tured in Dulbecco’s Modified Eagle’s Medium (DMEM, Merk, Cat. No. 30-2002, USA) supplemented with 10% fetal bovine serum (FBS, Merk, Cat. No. 16000044, USA) and 1% penicillin-streptomycin (Merk, Cat. No. 15140122, USA). Cells were maintained at 37°C in a hu midified incubator with 5% CO2. The cytotoxic effects of the treatments on breast cancer cells were evaluated using the (3-( 4 , 5 -dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) (Cat. No. E-CK-A341, ELabScience, China). Cells were seeded in 96-well plates at a density of 5×10³ cells per well and allowed to adhere overnight. Subsequently, cells were treated for 48 hours under the following conditions: i. Control (vehicle) ii. DMBA (80 μg/mL) iii. DMBA+cobalt chloride (80 μg/mL+50 μg/mL) iv. DMBA+zinc chloride (80 μg/mL+50 μg/mL) v. DMBA+cadmium nitrate (80 μg/mL+100 ng/mL) The concentrations of DMBA and trace elements were based on prior studies that demonstrating their biological effects in rodent models ( 10 , 21 - 23 ). After treatment, MTT solution (5 mg/mL, Cat. No. E-CK-A341, ELabScience, China) was added to each well, and plates were incubated for 4 hours. Formazan crystals were then solubilized with dimethyl sulfoxide (DMSO, Cat. No. 472301, Sigma Aldrich, USA), and absorbance was measured at 570 nm using a microplate reader (96 well, ELabScience, China). Cell viability was calculated as a percentage relative to the control group. All experiments were performed in 10 replicates, and results were presented as mean ± standard deviation (SD). Numerical data were analyzed using the Statistical Package for the Social Science (SPSS) version 26.0 (IBM, USA). Data are presented as mean ± standard deviation (SD). Group means were compared using one-way analysis of variance (ANOVA), followed by the Least Significant Differences (LSD) post hoc test. Statistical significance was set at P≤0.05. Prior to ANOVA, the normality of data distribution for each group was assessed using the Shapiro-Wilk test.