Plasticisers, such as Di(2-ethylhexyl) phthalate (DEHP), are found  in many  polyvinyl chloride (PVC) products (An et al., 2024; Zöngür, 2024) and can migrate into the environment and then into the human body through either oral ingestion or  inhalation, and dermal contact (Ding, S., Qi, et al., 2021; Rowdhwal & Chen, 2018). After being absorbed, DEHP is quickly broken down in the liver and gut into Mono-Ethylhexylphthalate (MEHP) and other oxidative metabolites (Chen et al., 2022; Koch et al., 2005). The parent compound has a short half-life, but repeated exposure keeps the active metabolites in the body, which can cause cumulative toxicity in different organ systems (Rousseau-Ralliard et al., 2024; Aydemir et al., 2024; Bijjala et al., 2025). Chronic human exposure is significantly correlated with metabolic disorders, such as diabetes, hyperlipidemia, insulin resistance, and non-alcoholic fatty liver disease (NAFLD) (Robles-Matos et al., 2023; He et al., 2023; Carli et al., 2022).

 

The liver is the main site of DEHP metabolism and a major target for its toxicity (Singh, Pandey, et al., 2025). Mechanisms behind the liver damage induced by DEHP include an interaction among oxidative stress, mitochondrial dysfunction and inflammation (Behairy et al., 2021; Fang et al., 2025; Guo, Deng, et al., 2024; Zhang, Zhao, et al., 2022). More specifically, DEHP induced liver failure occurs through the activation of thioredoxin-interacting protein (TXNIP), which inhibits important transcriptional factors (PGC-1α, TFAM and NRF1) in mitochondrial biogenesis while activating the NLRP3 inflammasome (Liu, Han, et al., 2021; Xu et al., 2024). TXNIP-mediated signalling activates the NF-κB/MAPK pathway and generates ROS, inducing apoptosis and dysfunction in hepatocytes (Dagdeviren et al., 2023; Xu et al., 2024).

 

The manifestation of liver damage is evidently dose-dependent. Steatosis (lipid deposition) typically arises with moderate DEHP dosing, whereas high exposure results in significant cellular damage and necrosis, with

 

 

 

 

 

 

 

 

 

 

 

minimal lipid deposition (Robles-Matos et al., 2023), indicating a metabolic transition from lipid storage to oxidative stress (ROS)-induced injury (Wang et al., 2025; Yang et al., 2025). Furthermore, DEHP can provoke an inflammatory response in Kupffer cells, leading to elevated levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) that may contribute to cholestasis and bile duct damage via metabolic reprogramming (Yu et al., 2025; Wu, Zhang, et al., 2022).

 

DEHP causes kidney injuries through several mechanisms. DEHP triggers oxidative and inflammatory pathways to cause damage to the kidneys (Ding, Huang, et al., 2023), as well as cellular pathways that include stress kinase pathways and transcription factor pathways (Kandel et al., 2025). Exposure to DEHP leads to the activation of p38 MAPK and NF-κB signalling pathways in the kidney, causing the inflammation and destruction of podocytes (Shi et al., 2023). Another mechanism of  DEHP  injury to the kidneys is through the activation of the NLRP3 inflammasome and caspase-1, which drive a type of programmed cell death known as pyroptosis in renal tubular cells that is characterized by the cleavage of gasdermin-D (Li, Gu, et al., 2024; Wei et al., 2025).

 

The increase in the production of IL-1β, TNF-α, and caspase-1 in the kidneys due to exposure to DEHP are the indicators of damage caused by DEHP is due to the activation of the inflammasome (Aranda-Rivera et al., 2022; Wei et al., 2025). DEHP also causes intrinsic apoptosis in renal epithelial cells through an increase in the expression of pro-apoptotic proteins (Bax, caspase-3), whereas it has a decrease in the expression of anti-apoptotic protein (Bcl-2) (Maire et al., 2005; Ran et al., 2025). The histological manifestations of this molecular injury include tubular atrophy, glomerular hypertrophy, interstitial fibrosis, and leukocyte infiltration (Liu, Zhang, et al., 2025). Thus, DEHP's nephrotoxicity is due to the loss of the balance between ROS/Nrf2, activation of MAPK and NF-κB signalling pathways, NLRP3/caspase-1 induced pyroptosis and intrinsic apoptosis (Bax/Bcl-2/caspase-9/3) (Gad El-Karim et al., 2023; Singh, Jangra, & Kumar, 2024; Pan et al., 2025).

 

DEHP influences blood cell production by inducing an inflammatory environment in the bone marrow and disrupting redox balance thereby interfering with signalling pathways (Tsai et al., 2022; Hu et al., 2023). Inflammation and oxidative stress arise when DEHP-induced reactive oxygen species (ROS) stimulate the NF-κB pathway and disturb the normal cytokine equilibrium in the bone marrow (Kaiser et al., 2021; Lingappan, 2018). Oxidative stress and the consequent disturbance of cytokine equilibrium are associated with heightened apoptosis in bone marrow cells, reductions in specific leukocyte and lymphocyte populations, and elevations in neutrophils, ultimately resulting in alterations in peripheral blood counts (Chakraborty et al., 2023; Chi et al., 2024; Manz et al., 2015).

 

High dose DEHP is known to affect vascular endothelial growth factor (VEGF) signalling, and to induce an attenuation in the expression of genes related to erythropoiesis/myelopoiesis thereby reducing the number of hematopoietic stem cells (HSCs), and also reducing the circulating population of erythrocytes (Hu et al., 2023). Therefore the reduction in angiogenic support, and the induction of apoptosis in progenitor cells clearly represent a mechanistic explanation for DEHP-induced haematotoxicity (Hussein et al., 2023). Importantly, these mechanisms were noted at environmentally relevant doses and therefore it is imperative that there be a rapid and extensive evaluation of possible protective interventions (Soni et al., 2022).

 

Given that the cause of toxicity from exposure to DEHP would be due to oxidative stress, mitochondrial dysfunction, inflammation, pyroptosis/apoptosis, and a disruption in metabolism, there is rationale for using antioxidants, anti-inflammatory drugs, and mitochondrial protective compounds to either prevent or diminish the negative effects caused by exposure to DEHP.

 

Lactobacillus rhamnosus is an effective probiotic for its antioxidant and immunomodulatory effects (Steele, 2022; Rocha-Ramírez et al., 2021). In addition, oral administration of L. rhamnosus was demonstrated to be capable of enhancing antioxidant defenses in both the liver (Chen, Kong et al., 2022) and bone marrow (i.e., through increasing levels of MnSOD and AMPKα) as well as supporting mitochondrial function (López-Almada et al., 2024; Zhang, Sun, et al., 2024; Zhang, Wang, et al., 2015). L. rhamnosus GG restored redox balance and elevated Higd2a mRNA expression and MnSOD1 protein levels in both the liver and bone marrow of ethanol exposed rats (Salazar et al., 2025). Moreover, L. rhamnosus GG increases gut microbiota diversity and decreases endotoxemia indicating that it could possibly protect against systemic DEHP toxicity by way of the gut-liver axis and Nrf2 mediated signalling (Sun et al., 2025; Baralić et al., 2020; Zheng et al., 2022; Guo, Yu, et al., 2023).

Extracts of Tribulus terrestris fruit are a rich source of steroidal saponins and flavonoids(Saeed et al., 2024).  There are many studies that demonstrate Tribulus terrestris has significant hepatoprotective as well as nephroprotective properties (Mohy-Ud-Din & Jonassaint, 2024). Tribulus terrestris normalizes serum liver enzymes (AST and ALT), and serum creatine levels in toxicological  studies without any adverse effects being observed in tissue histopathology (Darbandi & Jabbar, 2025; Al-Mohamadi et al., 2025). The protective actions of Tribulus terrestris include restoring glutathione (GSH) levels, inhibiting lipid peroxidation, and inhibiting the NF-κB/IL-6 inflammatory signalling pathways (Shetty et al., 2024; Abdi et al., 2025; Chen, Deng, et al., 2025).

 

Vitamin C is a powerful antioxidant that can neutralize free radicals in the aqueous phase of the body and also acts to regenerate other antioxidants (e.g. Vitamin E) (Alberts et al., 2025). It has nearly completely restored DEHP-disrupted metabolic pathways, blood glucose and lipid profiles and depleted antioxidant enzyme activity, for instance, superoxide dismutase (SOD) (Manokaran et al., 2025; Soni et al., 2022; Mo et al., 2024). The reduction of reactive oxygen species (ROS) in plasma by Vitamin C, and its inhibition of oxidative DNA damage, and subsequent inflammatory cascades make Vitamin C a crucial first line of defense (Kawashima et al., 2015; Gęgotek & Skrzydlewska, 2022; Ibrahim et al., 2022).

Vitamin E and Selenium are synergistic when used together. Vitamin E protects the cell membranes from peroxidation and Selenium is essential for the glutathione peroxidase (GPx) that breaks down hydro peroxides (Kilicarslan You et al., 2024; Shahidin et al., 2025). Co-treatment with both Vitamins E and Selenium has been shown to greatly reduce DEHP-induced toxicity and normalize blood glucose levels and liver function tests, and decrease spleen damage more effectively than either treatment separately (Haque et al., 2025). In summary, they provide a comprehensive defence mechanism against oxidative injury (Ding, Sun, et al., 2025; Xiao et al., 2021) and can limit phthalate-induced harm” (Alam & Hoque, 2018).

 

Zinc and manganese are two essential trace minerals that serve as cofactors for several key antioxidant enzymes (El-Sayed et al., 2024). Zinc is required for Cu/Zn superoxide dismutase (SOD) activity and increase expression of metallothionein, which will bind free radicals (Hübner & Haase, 2021; Briassoulis et al., 2023). Manganese is necessary to have active mitochondrial Mn-SOD(SOD2) activity (Grujicic & Allen, 2025). The SOD/GPx enzyme system is replaced by Zn, Se, and Mn in detoxification (Manful et al., 2025). Therefore, supplementing Zn and Mn can also restore endogenous enzymatic defenses and reduce the amount of ROS produced at the mitochondria due to DEHP exposure (Oyovwi et al., 2025; Xia et al., 2024).

 

The hypothesis of this study is that the multiorgan oxidative stress caused by DEHP are due to overlap in pathways of oxidative stress, inflammation and metabolic alterations and its exposure can be significantly reduced using pharmacologic and nutritional interventions that target both enzymatic and non-enzymatic antioxidant defence mechanisms, inflammatory signalling pathways, mitochondrial energy production, and changes in the gut microflora to restore the mucosal barrier function. This comprehensive study compares the effectiveness of seven different protective treatments (L.rhamnosus, T. terrestris, Vitamins C and E, Se, Zn, Mn) when administered singly to compare their relative effectiveness and to determine those treatments that should be examined further as part of multi-modal treatment regimens including synergetic combinations of botanicals, probiotics and micronutrient based treatments.

 

Preparation of tissue homogenates

The liver and kidney tissue from each rat were placed in 10 ml/g ice-cold phosphate-buffer saline (pH 7.5) and homogenized using a glass homogenizer. The homogenates were then centrifuged at 10,000 g for 10 min at 4^oC, and the resultant supernatants were collected for later detection of biochemical parameters by following the under mentioned standard methodologies.

 

Haematological Analysis

For the haematological examination, Blood samples were collected from the retro orbital plexus of the rats using an heparinized capillary tube. The  haematological  analyses were measured with an auto cell counter for veterinary purposes, specific for rat (MS9-5 of Melet Schloesing Lab, New Delhi, India).

 

Serum Biochemical Parameters

For biochemical investigations serum was separated by centrifugation at 4000 rpm for 10 minutes and kept at -4 °C. Serum biochemical analyses were conducted using standard colorimetric and enzymatic methods on an automated biochemistry analyzer (Hitachi 912, Roche, Switzerland):

 

Renal Function Markers: Serum creatinine (mg/dl): serum creatinine was estimated by alkaline Jaffe’s Picrate method and Blood urea nitrogen (BUN, mg/dl) estimated by enzymatic urease method

All analyses were performed using commercially available diagnostic kits  according to manufacturer’s instructions with appropriate quality controls. Absorbance measurements were obtained at appropriate wavelengths using a spectrophotometer (Hitachi U-2800) with temperature control at 37°C.

 

Statistical Analysis

All data were expressed as mean ± standard deviation (SD) for six animals per group. Statistical significance between groups was evaluated using one-way analysis of variance (ANOVA), followed by Duncan’s Multiple Range Test (DMRT). Differences were considered significant at p<0.05, very significant at p<0.01, and highly significant at p<0.001. Percent deviation over control (PDC) and percent deviation over DEHP-treated values (PDE) were calculated for comparative interpretation.

Percent Deviation from Control (PDC) was calculated for each parameter as:

 PDC (%) = [(Treated group mean - Control group mean) / Control group mean] × 100

Percent Deviation from DEHP (PDE) was calculated for each treated group as:

PDE (%) = [(Treated group mean - DEHP group mean) / DEHP group mean] × 100

Percent Recovery (PR) was calculated for each parameter using the formula:

PR (%) = [(Treated group mean - DEHP group mean) / (Control group mean - DEHP group mean)] × 100

 

Growth Performance and Metabolic Parameters

Compared to the Control Group (T1), DEHP treated rats (T2) had statistically significant depressed performance in body weight gain, which averaged 30.35 % less than the Control group (Control = 254.34 ± 7.06 g; DEHP = 177.15 ± 4.91 g, p < 0.001). Rats exposed to DEHP experienced severe suppression in weight gain compared to Controls (Loss in DEHP = 47.19 ± 1.31 g; Gain in Controls = 31.08 ± 0.86 g); this represented a 51.83 % deviation below that of the Controls. As shown in Table 1, food intake in DEHP-treated rats decreased by 25.83 % to 15.22 ± 0.42 g / 100 g bw / day; water consumption decreased by 28.76 % to 22.17 ± 0.61 mL / rat / day; and urine production decreased by 32.20 % to 4.19 ± 0.12 mL / rat / day, all of which were significantly (p < 0.001) reduced relative to the Controls.

As illustrated in Table 1, Tribulus terrestris (T3) provided a superior degree of restoration of growth related parameters to the baseline level (70.88 % restoration of final body weight = 231.86 ± 6.42 g; 70.45 % restoration of weight gain / loss; 73.96 % restoration of food intake; and 70.95 % restoration of water consumption). The restoration of growth related parameters was slightly less than T3 in the Lactobacillus rhamnosus (T4) group (62.29 % restoration of final body weight; 66.29 % restoration of composite metabolic parameters). Vitamin E (T8) restored 53.48 % of final body weight. The supplemental trace minerals (T5-T7) exhibited progressive decreases in restoration capability, with the manganese supplement (T7) exhibiting only 30.52 % restoration of final body weight. In addition to final body weight, the restoration of water and urine parameters was consistent with the pattern observed with growth related parameters.

 

Antioxidative Defense Mechanisms In The Liver

The liver's defense mechanisms against oxidative stress were severely affected by DEHP exposure; SOD activity was significantly reduced (by 45%) to 34.19 ± 0.95 U/mg protein (p < 0.001); catalase activity was reduced by 47% to 46.61 ± 1.30 µ moles of H₂O₂ degraded/mg protein/min; GPx activity was increased (by 43%) to 26.20 ± 0.73 µ moles of NADPH oxidized / mg protein / min. Glutathione-S-transferase activity was also increased (by 46%) to 7.90 ± 0.22 µ moles of thioether formed/ mg of protein/min. However, reduced glutathione (GSH) levels were significantly reduced (to 53%) to 7.670 ± 0.213 µ moles/gm wet weight of the tissue; glutathione reductase activity was significantly reduced (by 43%) to 3.560 ± 0.099 units/mg protein (p<0.001).

Treatment with Tribulus terrestris resulted in the most complete recovery of the liver's antioxidative balance as evidenced by an increase of 69.9% in SOD activity, 73.1% in CAT activity, 74.8% in GPX activity, and 71.39% in GSH levels. The recovery values for SOD activity for L. rhamnosus (66.6%), CAT activity for L. rhamnosus (67.5%), GPx activity for L. rhamnosus (68.7%) and GSH levels for L. rhamnosus (68.4%) were very similar to those for Tribulus terrestris, although somewhat lower in each case. The recovery values for SOD activity for vitamin E (54.2%) and vitamin C (40.1%) were both less than that of Tribulus terrestris and L. rhamnosus. Individually administered trace elements had varying effects on the recovery of liver antioxidative balance with selenium resulting in a 38.5% recovery of SOD activity, zinc a 30.8% recovery of SOD activity, and manganese only a 25.2% recovery of SOD activity.

 

Mitochondrial Function And Energy Metabolic Enzymes of the Liver

Hepatic mitochondria are involved in the synthesis of ATP and other high-energy compounds through the process of cellular respiration. DEHP exposure was found to have profoundly impacted the ability of these organelles to perform their functions. Specifically, glucose-6-phosphate dehydrogenase (G6PDH) activity was increased (by 63.4%) to 12.79 ± 0.36 µ moles of formazan formed/mg protein/min (p<0.001), indicating a compensatory mechanism to help restore cellular energy status. Succinate dehydrogenase (SDH) activity was decreased (by 52%) to 1.41 ± 0.04 (p < 0.001); pyruvate dehydrogenase (PDH) activity was decreased (by 45%) to 1.29 ± 0.04; lactate dehydrogenase (LDH) activity was increased (by 41.9%) to 1.72 ± 0.05 (p<0.001). This suggests that oxidative phosphorylation is being disrupted and energy is being produced through anaerobic glycolysis rather than aerobic oxidation of carbohydrates.

 

Tribulus terrestris demonstrated the most significant effect on restoring mitochondrial and energy metabolic enzymatic function of all treatments tested as indicated by its comparative efficiency index (CEI = 0.7021) which was the highest CEI observed among the treatments tested. Tribulus terrestris demonstrated 71.96% recovery of G6PDH activity, 71.61% recovery of SDH activity, 71.85% recovery of PDH activity, and 69.86% recovery of LDH activity. L. rhamnosus demonstrated recoveries of 65.31% for G6PDH activity and 64.17% for SDH activity. Vitamin E demonstrated a moderate level of efficacy (CEI = 0.5457) in terms of restoring mitochondrial and energy metabolic enzymatic function of the liver. Manganese demonstrated the least effective treatment (CEI = 0.2998) in this regard.

 

Hepatic Marker Enzymes and Liver Function

AST was increased to 122.67±3.41 μmoles pyruvate/g wet tissue/min (a 47.00% increase above control; 83.45±2.32; p<0.001). The level of ALT was increased to 58.18±1.62, suggesting severe liver damage through cytoplasmic enzyme release by the damaged hepatocytes. The elevated levels of both enzymes in addition to the increases in ALP (to 7.52±0.21 µmoles of p-nitrophenyl formed / gram wet weight of tissue / min, a 46.02% elevation) and ACP (to 16.15±0.45, a 44.97% elevation over the control value of 11.14±0.31) provide evidence for significant disruption of liver function and morphology (Table 6).

The Tribulus terrestris treated group had a 96.11±2.67 AST recovery (a 67.72% recovery); 45.69±1.27 ALT recovery (a 69.18% recovery); 12.60±0.35 ACP recovery (a 70.86% recovery); and 5.92±0.16 ALP recovery (a 67.51% recovery). In comparison, Lactobacillus rhamnosus had a 60.17% AST recovery and a 65.72% ALT recovery. Vitamin E also demonstrated some degree of recovery, but this was less than that observed for Tribulus terrestris (57.09% AST recovery), and individual trace elements also demonstrated varying degrees of efficacy. Zinc provided the least amount of recovery (35.77% AST recovery), whereas selenium provided a 45.08% AST recovery and manganese only a 28.61% AST recovery.

 

Renal Antioxidant Enzyme Status

Antioxidant enzymes within the kidney exhibited suppressed activities similar to those seen in the liver. Renal superoxide dismutase (SOD) activity was decreased to 25.72±0.72 units/mg protein (a 43.02% decrease from the control value of 45.14±1.26, p<0.001). Catalase activity was decreased to 35.85±1.00 (a 45.00% decrease from the control value of 65.19±1.81), and glutathione-S-transferase (GST) activity was decreased to 2.09±0.06 (a 44.11% decrease from the control value of 3.74±0.10). Glutathione peroxidase (GPx) activity, however, was increased to 18.04±0.50 (a 45.01% increase over the control value of 12.44±0.35). Glutathione (GSH) was reduced to 3.948±0.110 Reduced Glutathione (GSH)- µ moles/gm wet weight of the tissue (a 47.01% decrease from the control value of 7.450±0.207), and glutathione reductase activity was increased to 18.038±0.502 units/mg protein (a 45.00% increase over the control value of 12.445±0.347), indicating that there is an activation of the cellular response to oxidative stress, which does not appear sufficient to mitigate the toxic effects associated with DEHP exposure (Table 4).

 

The Tribulus terrestris treated group demonstrated complete recovery of renal antioxidant enzyme activity as evidenced by SOD activity being restored to 39.46±1.10 (a 70.75% restoration), catalase activity being restored to 56.02±1.56 (a 68.73% restoration), GST activity being restored to 3.25±0.09 (a 72.50% restoration), and GSH being restored to 6.443±0.179 (a 71.248% restoration). Similarly, Lactobacillus rhamnosus demonstrated recovery of 60.76% for SOD and 66.89% for catalase. The mean percent recovery (MPR) data indicate that Tribulus terrestris demonstrated an overall recovery of 70.21% for renal antioxidant enzyme activity when averaged across the four measured enzyme activities, compared to 62.77% for Lactobacillus rhamnosus and 54.57% for vitamin E.

 

Renal Mitochondrial and Energy Metabolism Enzymes

Mitochondrial enzyme activities within the kidney were similarly disrupted. G6PDH activity was increased to 6.760±0.188 µ moles of formazan formed/mg protein/min (a 42.92% increase over the control value of 4.730±0.132), succinate dehydrogenase (SDH) activity was decreased to 0.983±0.027 (a 47.01% decrease from the control value of 1.855±0.052), pyruvate dehydrogenase (PDH) activity was decreased to 1.010±0.028 (a 42.05% decrease from the control value of 1.743±0.048), lactate dehydrogenase (LDH) activity was increased to 1.254±0.035 (a 39.96% increase over the control value of 0.896±0.025) (Table 5). The Tribulus terrestris treated group demonstrated the greatest recovery of renal mitochondrial enzyme activities as evidenced by a 67.286% G6PDH recovery, a 69.487% SDH recovery, a 68.515% PDH recovery, and a 65.311% LDH recovery. The Lactobacillus rhamnosus treated group demonstrated comparable recovery with a 65.472% G6PDH recovery and a 66.697% SDH recovery. Selenium demonstrated the most effective supplementation effect with a 51.282% G6PDH recovery, while manganese provided the least recovery (a 27.439% G6PDH recovery).

 

Enzymes in the Renal Mitochondria for Energy Production:

The observed alterations in kidney mitochondria enzymes are consistent with those found in liver mitochondria. The activity of G6PDH was elevated by 42.92% to 6.760 ± 0.188 µ moles of formazan formed/mg protein/min compared to controls (4.730 ± 0.132); the activity of SDH was reduced by 47.01% to 0.983 ± 0.027 compared to controls (1.855 ± 0.052); the activity of PDH was reduced by 42.05% to 1.010 ± 0.028 compared to controls (1.743 ± 0.048); and the activity of LDH was elevated by 39.96% to 1.254 ± 0.035 compared to controls (0.896 ± 0.025). (Table 5). A higher degree of recovery of renal mitochondrial enzyme activities for G6PDH (67.286%), SDH (69.487%), PDH (68.515%) and LDH (65.311%) were observed for Tribulus terrestris than for L. rhamnosus which showed a similar recovery for G6PDH (65.472%) and SDH (66.697%). Variable efficacy of individual trace elements for the recovery of these enzymes was shown; e.g., selenium recovered 51.282% of G6PDH activity while manganese recovered only 27.439%.

 

Biomarkers of Function of the Kidney:

Renal functional biomarkers demonstrated significant disruptions in kidney function secondary to DEHP exposure. Serum creatinine was significantly increased to 1.38±0.038 mg/dl (a 165.38% increase from the control value of 0.520±0.014, p<0.001), and serum blood urea nitrogen (BUN) was significantly increased to 48.60±1.351 mg/dl (a 165.57% increase from the control value of 18.300±0.509). These increases demonstrate that kidney function is severely impaired due to DEHP exposure (Table 8).

 

The Tribulus terrestris treated group demonstrated the most extensive recovery of kidney function as evidenced by a 68.493% recovery of serum creatinine (a recovery to 0.791±0.022 mg/dl) and a 67.203% recovery of serum BUN (a recovery to 28.24±0.785 mg/dl), and these values represent the largest recoveries observed for any of the treatments. The  Lactobacillus rhamnosus  treated group demonstrated a 63.246% recovery of serum creatinine and a 61.101% recovery of serum BUN. Vitamin E demonstrated moderate kidney protection with a 52.174% recovery of serum creatinine and a 54.814% recovery of serum BUN. Zinc demonstrated the least efficacy with only a 30.261% recovery of serum creatinine.

Hematological Parameters and Blood Cell Indices

Hemoglobin (Hb) was decreased to 6.93±0.19 g/dl (a 39.00% decrease from the control value of 11.36±0.32, p<0.001), red blood corpuscles (RBC) was decreased to 5.09±0.14 ×10⁶/mm³ (a 38.97% decrease from the control value of 8.34±0.23), hematocrit (HCT) was decreased to 30.70±0.85% (a 37.00% decrease from the control value of 48.73±1.36), mean corpuscular hemoglobin (MCH) was decreased to 11.81±0.33 Hemoglobin amount/RBC (pg) (a 37.01% decrease from the control value of 18.75±0.52), mean corpuscular volume (MCV) was decreased to 51.00±1.42 Mean RBC cell size (fl) (a 29.01% decrease from the control value of 71.84±1.99), and white blood corpuscles (WBC) was decreased to 4.94±0.14 ×10⁹/L (a 33.06% decrease from the control value of 7.38±0.21). Neutrophils were decreased to 56.76±1.58 ×10⁸/L (a 27.99% decrease from the control value of 78.83±2.19), and lymphocytes were decreased to 14.77±0.41 ×10⁸/L (a 32.00% decrease from the control value of 21.72±0.60). The mean corpuscular hemoglobin concentration (MCHC) remained unchanged (a 2.36% decrease), indicating that there was a normocytic/normochromic anemia. (Table 7).

The Tribulus terrestris treated group demonstrated the most extensive recovery of hematologic values as evidenced by a 69.75% recovery of Hb (a recovery to 10.02±0.28 g/dl), a 71.69% recovery of RBC (a recovery to 7.42±0.21 ×10⁶/mm³), a 69.27% recovery of HCT (a recovery to 43.19±1.20%), a 70.61% recovery of MCH (a recovery to 16.71±0.46 Hemoglobin amount/RBC (pg)), and a 74.47% recovery of MCV (a recovery to 66.52±1.85 Mean RBC cell size (fl)). The mean percent recovery (MPR) data demonstrate that Tribulus terrestris recovered approximately 71.28% of the measured hematologic parameters. The Lactobacillus rhamnosus treated group demonstrated an MPR of 64.60%, and the vitamin E treated group demonstrated an MPR of 55.40%. The vitamin C treated group demonstrated the least efficacy with an MPR of 43.89%, while selenium demonstrated an MPR of 49.27%, and zinc demonstrated an MPR of 38.50%. Manganese demonstrated the least efficacy with an MPR of 31.08%. White blood cells demonstrated similar trends in recovery as evidenced by a 72.13% recovery of WBCs (a recovery to 6.70±0.19 ×10⁹/L), a 65.98% recovery of WBCs by the Lactobacillus rhamnosus treated group, and a 56.15% recovery of WBCs by the vitamin E treated group.

 

Comparative Analysis and Mean Percent Recovery (MPR) / Comparative Efficiency Index (CEI)

Comprehensive analysis across all eight analytical tables (growth performance, hepatic antioxidants, hepatic mitochondrial enzymes, hepatic marker enzymes, renal antioxidants, renal mitochondrial enzymes, hematological parameters, and renal functional biomarkers) yielded the following treatment efficacy ranking based on mean percent recovery:

 

 

 

 

 

 

 

1.      Abdi, M., Karimzadeh, H., Jourabchi, A., Seghinsara, A. M., & Khodaie, L. (2025). Protective effects of Tribulus terrestris extract on cisplatin-induced ovarian damage: Antioxidants and anti-inflammatory insights. Human & Experimental Toxicology, 44. https://doi.org/10.1177/09603271251353492

2.      Aebi, H. (1984). Catalase in vitro. Methods in Enzymology, 105, 121–126.

3.      Ahmed, H. M., Shehata, H. H., El-Saeed, G. S. M., Gabal, H. H. A., & El-Daly, S. M. (2022). Ameliorative effect of Lactobacillus rhamnosus GG on acetaminophen-induced hepatotoxicity via PKC/Nrf2/PGC-1α pathway. Journal of Genetic Engineering and Biotechnology, 20(1), 142. https://doi.org/10.1186/s43141-022-00422-4

4.      Akerboom, T. P. M., & Sies, H. (1981). Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods in Enzymology, 77, 373–382.

5.      Alam, M. S., & Hoque, M. N. (2018). Prophylactic effects of vitamin E and selenium on di-n-butyl phthalate-induced testicular damage in prepubertal rats. Journal of Advanced Biotechnology and Experimental Therapeutics. https://doi.org/10.5455/jabet.2018.d12

6.      Alberts, A., Moldoveanu, E.-T., Niculescu, A.-G., & Grumezescu, A. M. (2025). Vitamin C: A comprehensive review of its role in health, disease prevention, and therapeutic potential. Molecules, 30(3), 748. https://doi.org/10.3390/molecules30030748

7.      Al-Garadi, M. A., Al-Baadani, H. H., & Alqhtani, A. H. (2022). Growth performance, histological changes and functional tests of broiler chickens fed diets supplemented with Tribulus terrestris powder. Animals, 12(15), 1930. https://doi.org/10.3390/ani12151930

8.      Al-Mohamadi, A., Eldin, I. M. T., Ibrahim, D., & Albadani, R. (2025). Ameliorative effects of Tribulus terrestris Qutayb extracts against gentamicin-induced nephrotoxicity in Wistar rats. International Journal of Nephrology, 2025, 7620700. https://doi.org/10.1155/ijne/7620700

9.      An, J., Roh, H.-H., Jeong, H., Lee, K.-Y., & Rhim, T. (2024). Rapid assessment of di(2-ethylhexyl) phthalate migration from consumer PVC products. Toxics, 12(1), 7. https://doi.org/10.3390/toxics12010007

10.   Aranda-Rivera, A. K., Srivastava, A., Cruz-Gregorio, A., Pedraza-Chaverri, J., Mulay, S. R., & Scholze, A. (2022). Involvement of inflammasome components in kidney disease. Antioxidants, 11(2), 246. https://doi.org/10.3390/antiox11020246

11.   Aydemir, D., Karabulut, G., Barlas, N., & Ulusu, N. N. (2024). DEHP impairs the oxidative stress response and disrupts trace element and mineral metabolism within the mitochondria of detoxification organs. Toxicology and Industrial Health, 41, 1–14. https://doi.org/10.1177/07482337241306252

12.   Baralić, K., Živančević, K., Javorac, D., Buha Djordjevic, A., Anđelković, M., Jorgovanović, D., Antonijević Miljaković, E., Đurić, M., Bulat, Z., Antonijević, B., & Đukić-Ćosić, D. (2020). Multi-strain probiotic ameliorated toxic effects of phthalates and bisphenol A mixture in Wistar rats. Food and Chemical Toxicology, 143, 111540. https://doi.org/10.1016/j.fct.2020.111540

13.   Behairy, A., Abd El-Rahman, G. I., Aly, S. S. H., Fahmy, E. M., & Abd-Elhakim, Y. M. (2021). Di(2-ethylhexyl) adipate plasticizer triggers hepatic, brain, and cardiac injury in rats: Mitigating effect of Peganum harmala oil. Ecotoxicology and Environmental Safety, 208, 111620. https://doi.org/10.1016/j.ecoenv.2020.111620

14.   Bijjala, A. R., Poli, V., & Motireddy, S. R. (2025). Di-2-ethylhexyl phthalate (DEHP) toxicity: Organ-specific impacts and human-relevant findings in animal models & humans - Review. Journal of Toxicology and Risk Assessment, 10, 062. http://doi.org/10.23937/2572-4061.1510062

15.   Bodansky, A. (1933). Phosphatase studies. II. Determination of serum phosphatase. Factors influencing the accuracy of the determination. Journal of Biological Chemistry, 101, 93–104.

16.   Briassoulis, G., Briassoulis, P., Ilia, S., Miliaraki, M., & Briassouli, E. (2023). The anti-oxidative, anti-inflammatory, anti-apoptotic, and anti-necroptotic role of zinc in COVID-19 and sepsis. Antioxidants, 12(11), 1942. https://doi.org/10.3390/antiox12111942

17.   Carli, F., Ciociaro, D., & Gastaldelli, A. (2022). Assessment of exposure to di-(2-ethylhexyl) phthalate (DEHP) metabolites and bisphenol A (BPA) and its importance for the prevention of cardiometabolic diseases. Metabolites, 12(2), 167. https://doi.org/10.3390/metabo12020167

18.   Chakraborty, S., Tabrizi, Z., Bhatt, N. N., Franciosa, S. A., & Bracko, O. (2023). A brief overview of neutrophils in neurological diseases. Biomolecules, 13(5), 743. https://doi.org/10.3390/biom13050743

19.   Chen, L., Deng, Y., Hu, J., & Gong, X. (2025). Nephroprotective effects of substances of medicine food homology and traditional Chinese medicine phytochemicals against acute kidney injury. Frontiers in Pharmacology, 16, 1539886. https://doi.org/10.3389/fphar.2025.1539886

20.   Chen, L., Huang, Y. L., Liu, F., Huang, N., Zeng, D. C., Zhong, Y. B., Liao, J. H., & Wang, M. Y. (2025). Impact of di-2-ethylhexyl-phthalate on metabolic syndrome: Insights from network toxicology and molecular docking and dynamics. Diabetes, Metabolic Syndrome and Obesity, 18, 2277–2288. https://doi.org/10.2147/DMSO.S523668

21.   Chen, Q., Kong, Q., Tian, P., He, Y., Zhao, J., Zhang, H., Wang, G., & Chen, W. (2022). Lactic acid bacteria alleviate di-(2-ethylhexyl) phthalate-induced liver and testis toxicity via their bio-binding capacity, antioxidant capacity and regulation of the gut microbiota. Environmental Pollution, 305, 119197. https://doi.org/10.1016/j.envpol.2022.119197

22.   Chi, Z., Yang, H., & Liu, J. (2024). Study on the combined toxicity of DEHP and lead on the blood system of rats. Chemosphere, 349, 140908. https://doi.org/10.1016/j.chemosphere.2023.140908

23.   Chrétien, D., Pourrier, M., Bourgeron, T., et al. (1995). An improved spectrophotometric assay of pyruvate dehydrogenase in lactate dehydrogenase contaminated mitochondrial preparations from human skeletal muscle. Clinica Chimica Acta, 240(2), 129–136. https://doi.org/10.1016/0009-8981(95)06145-6

24.   Chromý, V., Rozkošná, K., & Sedlák, P. (2008). Determination of serum creatinine by Jaffe method and how to calibrate to eliminate matrix interference problems. Clinical Chemistry and Laboratory Medicine, 46(8), 1127–1133. https://doi.org/10.1515/CCLM.2008.224

25.   Dagdeviren, S., Lee, R. T., & Wu, N. (2023). Physiological and pathophysiological roles of thioredoxin interacting protein: A perspective on redox inflammation and metabolism. Antioxidants & Redox Signalling, 38(4-6), 442–460. https://doi.org/10.1089/ars.2022.0022

26.   Darbandi, N., & Jabbar, R. F. (2025). Tribulus terrestris L. hydroalcoholic extract revokes the hepatotoxicity induced by cytarabine in male Wistar rats. Pharmaceutical and Biomedical Research, 11(1), 13–22. https://doi.org/10.32598/PBR.11.1.1307.2

27.   David, R. M., Moore, M. R., Finney, D. C., & Guest, D. (2000). Chronic toxicity of di(2-ethylhexyl)phthalate in rats. Toxicological Sciences, 55(2), 433–443. https://doi.org/10.1093/toxsci/55.2.433

28.   Ding, S., Qi, W., Xu, Q., Zhao, T., Li, X., Yin, J., Zhang, R., Huo, C., Zhou, L., & Ye, L. (2021). Relationships between di-(2-ethylhexyl) phthalate exposure and lipid metabolism in adolescents: Human data and experimental rat model analyses. Environmental Pollution, 286, 117570. https://doi.org/10.1016/j.envpol.2021.117570

29.   Ding, W. J., Huang, S. L., Huang, S., Xu, W. P., & Wei, W. (2023). Di(2-ethylhexyl) phthalate mediates oxidative stress and activates p38MAPK/NF-kB to exacerbate diabetes-induced kidney injury in vitro and in vivo models. Toxicology Research, 12(2), 332–343. https://doi.org/10.1093/toxres/tfad022

30.   Ding, Y., Sun, R., Jiang, X., Hao, Y., Song, Y., Jia, X., Bai, Y., & Xia, C. (2025). Combined vitamin E and selenium supplementation enhances antioxidant status, reduces disease incidence, and improves economic returns in transition dairy cows. Veterinary World, 18(8), 2439–2449. https://doi.org/10.14202/vetworld.2025.2439-2449

31.   Ellman, G. L. (1959). Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics, 82(1), 70–77.

32.   El-Sayed, A., Ebissy, E., Mohamed, R., & Ateya, A. (2024). Effects of antioxidant vitamins (A, D, E) and trace elements (Cu, Mn, Se, Zn) administration on gene expression, metabolic, antioxidants and immunological profiles during transition period in dromedary camels. BMC Veterinary Research, 20(1), 101. https://doi.org/10.1186/s12917-024-03959-3

33.   Erkekoglu, P., Zeybek, N. D., Giray, B. K., Rachidi, W., Kızılgün, M., Hininger-Favier, I., Favier, A., Asan, E., & Hincal, F. (2014). The effects of di(2-ethylhexyl)phthalate on rat liver in relation to selenium status. International Journal of Experimental Pathology, 95(1), 64–77. https://doi.org/10.1111/iep.12059

34.   Fang, X., Wang, T., Lou, S., Qiao, Y., Zhang, B., & Zhou, L. (2025). Mechanisms of di(2-ethylhexyl) phthalate-induced lipid metabolism disorders: A review. Journal of Applied Toxicology, 45, 2463–2479. https://doi.org/10.1002/jat.4867

35.   Flohé, L., & Günzler, W. A. (1984). Assays of glutathione peroxidase. Methods in Enzymology, 105, 114–121.

36.   Gad El-Karim, D. R. S., Lebda, M. A., Alotaibi, B. S., El-Kott, A. F., Ghamry, H. I., & Shukry, M. (2023). Lutein modulates oxidative stress, inflammatory and apoptotic biomarkers related to di-2-ethylhexyl phthalate (DEHP) hepato-nephrotoxicity in male rats: Role of nuclear factor kappa B. Toxics, 11(9), 742. https://doi.org/10.3390/toxics11090742

37.   Grujicic, J., & Allen, A. R. (2025). Manganese superoxide dismutase: Structure, function, and implications in human disease. Antioxidants, 14(7), 848. https://doi.org/10.3390/antiox14070848

38.   Gülçin, İ. (2025). Antioxidants: A comprehensive review. Archives of Toxicology, 99, 1893–1997. https://doi.org/10.1007/s00204-025-03997-2

39.   Guo, H., Yu, L., Tian, F., Chen, W., & Zhai, Q. (2023). The potential therapeutic role of Lactobacillaceae rhamnosus for treatment of inflammatory bowel disease. Foods, 12(4), 692. https://doi.org/10.3390/foods12040692

40.   Guo, Q., Deng, T., Du, Y., Yao, W., Tian, W., Liao, H., Wang, Y., Li, J., Yan, W., & Li, Y. (2024). Impact of di-2-ethylhexyl phthalate on mitochondria-associated endoplasmic reticulum membranes and reproductive toxicity in ovary. Ecotoxicology and Environmental Safety, 282, 116679. https://doi.org/10.1016/j.ecoenv.2024.116679

41.   Habig, W. H., Pabst, M. J., & Jakoby, W. B. (1974). Glutathione S-transferases: The first enzymatic step in mercapturic acid formation. The Journal of Biological Chemistry, 249(22), 7130–7139.

42.   Haque, M. S., Mobarak, M. H., Basher, M. K., Sarkar, S., Sarker, S., & Islam, M. R. (2025). Ameliorative potential of vitamin E and selenium against di-2-ethylhexyl phthalate (DEHP)-induced toxicity in adult female mice. Veterinary Medicine International, 2025, 6844730. https://doi.org/10.1155/vmi/6844730

43.   He, Y., Zou, J., Hong, T., & Feng, D. (2023). Association between di-2-ethylhexyl phthalate and nonalcoholic fatty liver disease among US adults: Mediation analysis of body mass index and waist circumference in the NHANES. Food and Chemical Toxicology, 179, 113968. https://doi.org/10.1016/j.fct.2023.113968

44.   Hillmann, G. (1971). Continuation of the standard method of the determination of the acid phosphatase activity. Zeitschrift für Klinische Chemie und Klinische Biochemie, 9(4), 273–274.

45.   Hoffmann, A., Kleniewska, P., & Pawliczak, R. (2019). Antioxidative activity of probiotics. Archives of Medical Science, 17(3), 792–804. https://doi.org/10.5114/aoms.2019.89894

46.   Hosten, A. O. (1990). BUN and creatinine. In H. K. Walker, W. D. Hall, & J. W. Hurst (Eds.), Clinical Methods: The History, Physical, and Laboratory Examinations (3rd ed., Chapter 193). Butterworths. https://www.ncbi.nlm.nih.gov/books/NBK305/

47.   Hu, Y. X., Hu, B. W., Chen, Y. S., You, H. M., Bai, M. R., Zhang, L. J., Guo, Z. F., & Liang, C. (2023). Di-2-ethylhexyl phthalate impairs angiogenesis and hematopoiesis via suppressing VEGF signalling in zebrafish. Comparative Biochemistry and Physiology Part C: Toxicology & Pharmacology, 267, 109588. https://doi.org/10.1016/j.cbpc.2023.109588

48.   Hübner, C., & Haase, H. (2021). Interactions of zinc- and redox-signalling pathways. Redox Biology, 41, 101916. https://doi.org/10.1016/j.redox.2021.101916

49.   Hussein, A. M., Javad, S., & Zahra, S. (2023). Induction of caspase-dependent apoptosis in rat bone marrow mesenchymal stem cells due to di-2-ethylhexyl phthalate toxicity was found to arrest the cell cycle at the G1 stage. Current Stem Cell Research & Therapy, 18(8), 1106–1112. https://doi.org/10.2174/1574888X18666230106114727

50.   Ibrahim, N. N., Al-Mashhadane, F. A., & ALtaaye, S. K. A. I. (2022). Protective effects of vitamin C against oxidative stress induced by diethylene glycol in liver and kidney. International Journal of Health Sciences, 6(S5), 7077–7087. https://doi.org/10.53730/ijhs.v6nS5.11594

51.   Kaiser, L., Quint, I., Csuk, R., Jung, M., & Deigner, H. P. (2021). Lineage-selective disturbance of early human hematopoietic progenitor cell differentiation by the commonly used plasticizer di-2-ethylhexyl phthalate via reactive oxygen species: Fatty acid oxidation makes the difference. Cells, 10(10), 2703. https://doi.org/10.3390/cells10102703

52.   Kamboj, P., Aggarwal, M., Puri, S., & Singla, S. K. (2011). Effect of aqueous extract of Tribulus terrestris on oxalate-induced oxidative stress in rats. Indian Journal of Nephrology, 21(3), 154–159. https://doi.org/10.4103/0971-4065.83727

53.   Kandel, R., Roy, P., & Singh, K. P. (2025). Molecular basis of oxidative stress-induced acute kidney injury, kidney fibrosis, chronic kidney disease, and clinical significance of targeting reactive oxygen species-regulated pathways to treat kidney disease. Frontiers in Bioscience-Scholar, 17(3), 38963. https://doi.org/10.31083/FBS38963

54.   Kawashima, A., Sekizawa, A., Koide, K., Hasegawa, J., Satoh, K., Arakaki, T., Takenaka, S., & Matsuoka, R. (2015). Vitamin C induces the reduction of oxidative stress and paradoxically stimulates the apoptotic gene expression in extravillous trophoblasts derived from first-trimester tissue. Reproductive Sciences, 22(7), 783–790. https://doi.org/10.1177/1933719114561561

55.   Kilicarslan You, D., Fuwad, A., Lee, K. H., Kim, H. K., Kang, L., Kim, S. M., & Jeon, T.-J. (2024). Evaluation of the protective role of vitamin E against ROS-driven lipid oxidation in model cell membranes. Antioxidants, 13(9), 1135. https://doi.org/10.3390/antiox13091135

56.   Koch, H. M., Bolt, H. M., Preuss, R., & Angerer, J. (2005). New metabolites of di-(2-ethylhexyl) phthalate (DEHP) in human urine and serum after single oral doses of deuterium-labelled DEHP. Archives of Toxicology, 79(6), 367–376. https://doi.org/10.1007/s00204-004-0642-4

57.   Li, M.-Z., Zhao, Y., Dai, X.-Y., Talukder, M., & Li, J.-L. (2023). Lycopene ameliorates DEHP exposure-induced renal pyroptosis through the Nrf2/Keap-1/NLRP3/caspase-1 axis. The Journal of Nutritional Biochemistry, 113, 109266. https://doi.org/10.1016/j.jnutbio.2022.109266

58.   Li, S., Gu, X., Zhang, M., Jiang, Q., & Xu, T. (2024). Di(2-ethylhexyl) phthalate and polystyrene microplastics co-exposure caused oxidative stress to activate NF-κB/NLRP3 pathway aggravated pyroptosis and inflammation in mouse kidney. The Science of the Total Environment, 926, 171817. https://doi.org/10.1016/j.scitotenv.2024.171817

59.   Lingappan, K. (2018). NF-κB in oxidative stress. Current Opinion in Toxicology, 7, 81–86. https://doi.org/10.1016/j.cotox.2017.11.002

60.   Liu, H., Han, W., Zhu, S., Li, Z., & Liu, C. (2021). Effect of DEHP and DnOP on mitochondrial damage and related pathways of Nrf2 and SIRT1/PGC-1α in HepG2 cells. Food and Chemical Toxicology, 158, 112696. https://doi.org/10.1016/j.fct.2021.112696

61.   Liu, Y., Zhang, X., Yi, R., Tian, Q., Xu, J., Yan, X., Ma, J., Wang, S., & Yang, G. (2025). Exploring the nephrotoxicity and molecular mechanisms of di-2-ethylhexyl phthalate: A comprehensive review. Chemico-Biological Interactions, 405, 111310. https://doi.org/10.1016/j.cbi.2024.111310

62.   Löhr, G. W., & Waller, H. D. (1965). Glucose-6-phosphate dehydrogenase (Zwischenferment). In H. U. Bergmeyer (Ed.), Methods of Enzymatic Analysis (pp. 744–745). Academic Press.

63.   López-Almada, G., Mejía-León, M. E., & Salazar-López, N. J. (2024). Probiotic, postbiotic, and paraprobiotic effects of Lactobacillus rhamnosus as a modulator of obesity-associated factors. Foods, 13(22), 3529. https://doi.org/10.3390/foods13223529

64.   Ma, J., Li, Y., Li, Y., Bi, J., Liu, J., Li, J., Meng, F., Yan, L., Wen, J., Wang, X., Qin, J., Wang, S., & Du, R. (2026). Protective mechanisms of zinc and/or selenium supplementation against BPA-induced male and offspring reproductive toxicity: Insights from multiomics analysis. Environment International, 207, 109975. https://doi.org/10.1016/j.envint.2025.109975

65.   Maire, M. A., Rast, C., & Vasseur, P. (2005). Di-2-ethylhexylphthalate (DEHP) increases Bcl-2/Bax ratio and modifies c-myc expression in Syrian hamster embryo (SHE) cells. Toxicology Letters, 158(3), 237–245. https://doi.org/10.1016/j.toxlet.2005.04.004

66.   Manful, C. F., Fordjour, E., Subramaniam, D., Sey, A. A., Abbey, L., & Thomas, R. (2025). Antioxidants and reactive oxygen species: Shaping human health and disease outcomes. International Journal of Molecular Sciences, 26(15), 7520. https://doi.org/10.3390/ijms26157520

67.   Manokaran, K., Bhaskaran, R. S., Mudgal, J., Paramasivam, P., Shetty, S., Nayak, D., Carnelio, S., Vennila, J., Shanmugam, D., & Pai, S. K. (2025). Ascorbic acid alleviates reproductive toxicity of di-2-ethyl hexyl phthalate in female Wistar rats. Asian Pacific Journal of Reproduction, 14, 27–37. https://doi.org/10.4103/apjr.apjr_68_24

68.   Manz, P., Cadeddu, R.-P., Wilk, C., Fischer, J., Fritz, B., Haas, R., & Wenzel, F. (2015). Influence of di(2-ethylhexyl) phthalate on migration rate and differentiation of human hematopoietic stem and progenitor cells (CD34+). Clinical Hemorheology and Microcirculation, 61(4), 481–494. https://doi.org/10.3233/CH-151984

69.   Misra, H. P., & Fridovich, I. (1972). The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. The Journal of Biological Chemistry, 247(10), 3170–3175.

70.   Mo, H.-Y., Shan, C.-H., Chen, L.-W., Chen, X., Han, C., Wu, D., Tao, F.-B., & Gao, H. (2024). Antioxidant vitamins modification of the adverse health effects induced by phthalate exposure: A scoping review of epidemiological and experimental studies. Ecotoxicology and Environmental Safety, 286, 117190.

71.   Mohy-Ud-Din, N., & Jonassaint, N. (2024). Severe liver and renal injury from Tribulus terrestris. ACG Case Reports Journal, 11(2), e01267. https://doi.org/10.14309/crj.0000000000001267

72.   Mubarik, F., Khalil, A. A., Akhtar, M., Khalid, A., Basharat, S., Farooq, F., & Siddiqa, A. (2022). Assessing the antioxidant characteristics and polyphenol content of Tribulus terrestris microwave-assisted fruit extract. Phytopharmacology Research Journal, 1, 1–10.

73.   Nachlas, M. M., Margulius, S. P., & Seligman, A. M. (1960). A colorimetric method for the estimation of succinic dehydrogenase activity. Journal of Biological Chemistry, 235, 499–503.

74.   Oyovwi, M. O., Oyelami, J. O., Babawale, K. H., et al. (2025). Preventive antioxidant strategies to reduce the effects of phthalate exposure on reproductive health. Discover Environment, 3, 76. https://doi.org/10.1007/s44274-025-00257-z

75.   Pan, X., Zhu, R., Pei, J., & Zhang, L. (2025). Lycopene: A potent antioxidant to alleviate kidney disease. International Immunopharmacology, 151,114363. https://doi.org/10.1016/j.intimp.2025.114363

76.   Popli, S., Agarwal, T., Mishra, V., Kumar, A., & Saravanan, C. (2023). Adsorption characterization of Lactobacillus sp. for di-2-ethylhexyl phthalate. Probiotics and Antimicrobial Proteins, 16, 10055. https://doi.org/10.1007/s12602-023-10055-9

77.   Rahim, S., & Al-Nahi, A. (2024). Role of Tribulus terrestris as an antioxidant against oxidative stress induced by sorafenib in adult male rats. BIO Web of Conferences, 139, 06003. https://doi.org/10.1051/bioconf/202413906003

78.   Ran, B., Wang, X., Liu, B., Ye, J., Liu, L., Yin, Z., & Huang, Z. (2025). Toxicity and mechanistic analysis of di(2-ethylhexyl)phthalate in renal cell carcinoma progression: A systematic study with network toxicology and molecular docking strategies. Discover Oncology, 16(1), 1853. https://doi.org/10.1007/s12672-025-03543-7

79.   Reitman, S., & Frankel, S. (1957). A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases. American Journal of Clinical Pathology, 28(1), 56–63.

80.   Robles-Matos, N., Radaelli, E., Simmons, R. A., & Bartolomei, M. S. (2023). Preconception and developmental DEHP exposure alter liver metabolism in a sex-dependent manner in adult mouse offspring. Toxicology, 499, 153640. https://doi.org/10.1016/j.tox.2023.153640

81.   Rocha-Ramírez, L. M., Hernández-Chiañas, U., Moreno-Guerrero, S. S., Ramírez-Pacheco, A., & Eslava, C. A. (2021). Probiotic properties and immunomodulatory activity of Lactobacillus strains isolated from dairy products. Microorganisms, 9(4), 825. https://doi.org/10.3390/microorganisms9040825

82.   Rousseau-Ralliard, D., Bozec, J., Ouidir, M., Jovanovic, N., Gayrard, V., Mellouk, N., Dieudonné, M.-N., Picard-Hagen, N., Flores-Sanabria, M.-J., Jammes, H., Philippat, C., & Couturier-Tarrade, A. (2024). Short-half-life chemicals: Maternal exposure and offspring health consequences—The case of synthetic phenols, parabens, and phthalates. Toxics, 12(10), 710. https://doi.org/10.3390/toxics12100710

83.   Rowdhwal, S. S. S., & Chen, J. (2018). Toxic effects of di-2-ethylhexyl phthalate: An overview. BioMed Research International, 2018, 1750368. https://doi.org/10.1155/2018/1750368

84.   Saeed, M., Munawar, M., Bi Bi, J., Ahmed, S., Ahmad, M. Z., Kamboh, A. A., Arain, M. A., Naveed, M., & Chen, H. (2024). Promising phytopharmacology, nutritional potential, health benefits, and traditional usage of Tribulus terrestris L. herb. Heliyon, 10(4), e25549. https://doi.org/10.1016/j.heliyon.2024.e25549

85.   Sajedi, N., Ardakani, M. R., Madani, H., Naderi, A., & Miransari, M. (2011). The effects of selenium and other micronutrients on the antioxidant activities and yield of corn (Zea mays L.) under drought stress. Physiology and Molecular Biology of Plants, 17(3), 215–222. https://doi.org/10.1007/s12298-011-0067-5

86.   Salazar, C., Barreto, M., Adriasola-Carrasco, A. A., Carvajal, F., Lerma-Cabrera, J. M., & Ruiz, L. M. (2025). Lactobacillus rhamnosus GG modulates mitochondrial function and antioxidant responses in an ethanol-exposed in vivo model: Evidence of HIGD2A-dependent OXPHOS remodeling in the liver. Antioxidants, 14(6), 627. https://doi.org/10.3390/antiox14060627

87.   Shahidin, Wang, Y., Wu, Y., Chen, T., Wu, X., Yuan, W., Zhu, Q., Wang, X., & Zi, C. (2025). Selenium and selenoproteins: Mechanisms, health functions, and emerging applications. Molecules, 30(3), 437. https://doi.org/10.3390/molecules30030437

88.   Shetty, N., Holla, S., Nayak, V., Shenoy, V. B., & Mohandas, R. K. (2024). Antioxidant and anti-inflammatory activity by modulating IL-6 as a potential mechanism in the nephroprotective and hepatoprotective properties of Tribulus terrestris. Research in Pharmaceutical Sciences, 19(4), 376–386. https://doi.org/10.4103/RPS.RPS_66_23

89.   Shi, H., Zhao, X., Qin, P., Zhou, X., Liu, S., Sun, C., Cao, Q., Zhu, S., & Sun, S. (2023). Green tea polyphenols alleviate kidney injury induced by di-2-ethylhexyl phthalate in mice. American Journal of Nephrology, 55(1), 1–11. https://doi.org/10.1159/000534106

90.   Singh, L. K., Pandey, R., Siddiqi, N. J., & Sharma, B. (2025). Molecular mechanisms of phthalate-induced hepatic injury and amelioration by plant-based principles. Toxics, 13(1), 32. https://doi.org/10.3390/toxics13010032

91.   Soni, M., Shah, M. Z. U. H., & Shrivastava, V. K. (2022). Vitamin C mitigates hematological and biochemical alterations caused by di(2-ethylhexyl) phthalate toxicity in female albino mice, Mus musculus. Comparative Clinical Pathology, 31(6), 1005–1016. https://doi.org/10.1007/s00580-022-03400-x

92.   Steele, C. (2022). Lactobacillus rhamnosus GG: A review of clinical use and efficacy. Nutrition Medicine Journal, 13, 70–116.

93.   Sun, C., Zhu, J., Sun, X., Zhang, Z., Sun, Y., Jin, Y., & Wu, T. (2025). Targeting the human gut microbiome: A comparative review of probiotics, prebiotics, synbiotics, and postbiotics. Journal of Advanced Research. https://doi.org/10.1016/j.jare.2025.12.032

94.   Tsai, C. Y., Fang, T. P., Chen, S. W., Chen, H. W., Lin, E. C., Lin, T. A., Tarng, D. C., & Chang, Y. I. (2022). Di(2-ethylhexyl)phthalate impairs erythropoiesis via inducing Klotho expression and not via bioenergetic reprogramming. American Journal of Translational Research, 14(2), 1234–1245.

95.   Wang, K., Wang, Z., Ma, C., Yang, J., Xing, S., Song, B., Guo, C., Song, W., Cao, T., Bai, M., & Wang, Y. (2025). Mitochondria: A key regulator of programmed cell death in OP. Frontiers in Endocrinology, 16, 1576597. https://doi.org/10.3389/fendo.2025.1576597

96.   Wei, Y., Zhang, M., Song, J., Wang, T., Ma, Y., Qin, L., Li, J., Qian, X., & Chen, J. (2025). Nephrotoxicity of phthalates: A review based on epidemiological and toxicological evidence. Toxics, 13(11), 947. https://doi.org/10.3390/toxics13110947

97.   Xia, L. Z., Liu, L. L., Yue, J. Z., Lu, Z. Y., Deng, R. Y., He, X., Li, C. C., Hu, B., & Gao, H. T. (2024). Ameliorative effects of zinc and vitamin E against phthalates-induced reproductive toxicity in male rats. Environmental Toxicology, 39(6), 3330–3340. https://doi.org/10.1002/tox.24191

98.   Xiao, J., Khan, M. Z., Ma, Y., Alugongo, G. M., Ma, J., Chen, T., Khan, A., & Cao, Z. (2021). The antioxidant properties of selenium and vitamin E: Their role in periparturient dairy cattle health regulation. Antioxidants, 10(10), 1555. https://doi.org/10.3390/antiox10101555

99.   Xu, P., Su, Y.-N., Ling, C., Wang, J., & Zhang, W. (2024). Mitochondrial dysfunction mediated by thioredoxin-interacting protein: A crucial determinant in di-2-ethylhexyl phthalate-induced liver failure. Ecotoxicology and Environmental Safety, 272, 116103. https://doi.org/10.1016/j.ecoenv.2024.116103

100. Yang, X.-H., Cao, S.-L., Xu, J.-X., Zhang, G., Zhang, Y.-J., & Wang, Q.-N. (2025). Di(2-ethylhexyl) phthalate exposure alters hepatic lipid metabolism in male SAMP8 aged mice. Journal of Applied Toxicology. https://doi.org/10.1002/jat.4927

101. Yu, Y., Zhang, L., Zhou, J., Wang, J., & Huang, Q. (2025). Di(2-ethylhexyl) phthalate induces cholestasis liver injury in mice associated with promoting macrophage polarization through NRF2 signalling pathway. Toxicology Research, 14(6), tfaf151. https://doi.org/10.1093/toxres/tfaf151

102. Zhang, F., Zhen, H., Cheng, H., Hu, F., Jia, Y., Huang, B., & Jiang, M. (2022). Di-2-ethylhexyl phthalate exposure induces liver injury by promoting ferroptosis via downregulation of GPX4 in pregnant mice. Frontiers in Cell and Developmental Biology, 10, 1014243. https://doi.org/10.3389/fcell.2022.1014243

103. Zhang, H., Zhao, Y., Cui, J.-G., Li, X.-N., & Li, J.-L. (2022). DEHP-induced mitophagy and mitochondrial damage in the heart are associated with dysregulated mitochondrial biogenesis. Food and Chemical Toxicology, 161, 112818. https://doi.org/10.1016/j.fct.2022.112818

104. Zhang, M., Wang, C., Wang, C., Zhao, H., Zhao, C., Chen, Y., Wang, Y., McClain, C., & Feng, W. (2015). Enhanced AMPK phosphorylation contributes to the beneficial effects of Lactobacillus rhamnosus GG supernatant on chronic-alcohol-induced fatty liver disease. The Journal of Nutritional Biochemistry, 26(4), 337–344. https://doi.org/10.1016/j.jnutbio.2014.10.016

105. Zhang, X., Sun, L., Wu, M., Yu, C., Zhao, D., Wang, L., Zhang, Z., Yi, D., Hou, Y., & Wu, T. (2024). Effect of supplementation with Lactobacillus rhamnosus GG powder on intestinal and liver damage in broiler chickens challenged by lipopolysaccharide. Frontiers in Microbiology, 15, 1466274. https://doi.org/10.3389/fmicb.2024.1466274

106. Zheng, J., Ahmad, A. A., Yang, Y., Liang, Z., Shen, W., Feng, M., Shen, J., Lan, X., & Ding, X. (2022). Lactobacillus rhamnosus CY12 enhances intestinal barrier function by regulating tight junction protein expression, oxidative stress, and inflammation response in lipopolysaccharide-induced Caco-2 cells. International Journal of Molecular Sciences, 23(19), 11162. https://doi.org/10.3390/ijms231911162

 

107.          Zöngür, A. (2024). Evaluation of the effects of di-(2-ethylhexyl) phthalate (DEHP) on Caenorhabditis elegans survival and fertility. Applied Biochemistry and Biotechnology, 196(12), 8998–9009. https://doi.org/10.1007/s12010-024-05032-z