Gallic acid counteracts tartrazine-induced testicular dysfunction in rats: biochemical, histopathological and ultrastructural evidences

Abstract

Background

Tartrazine (Tz) is one of the most commonly used colorants incorporated in the food manufacturing. Its toxicity is derived from metabolic byproducts representing health hazards to consumers. Gallic acid (GA) is known for its redox stabilizing, anti-apoptotic, and cytoprotective characteristics. Therefore, the aim of this study is to explore the possible defensive effect of GA against Tz-induced testicular dysfunction. To achieve this objective, 18 male Wistar adult rats were randomly and equally categorized into three groups for 30 days. The control group received no treatment. Tz at a dose of 30 mg/kg BW was administered to the Tz group. The Tz + GA group received GA at a dose of 200 mg/kg BW in concurrent with the previously described Tz dosage. Both Tz and GA were supplemented orally once daily by a stomach tube.

Results

The marked decline in luteinizing hormone, follicle stimulating hormone, testosterone, and estradiol 17beta confirmed deviation in pituitary–gonadal axis of Tz-exposed rats. Imbalances in plasma redox equilibrium were evident, characterized by a notable increase in malondialdehyde and nitric oxide levels, along with a decrease in reduced glutathione and total antioxidant capacity. Deteriorations in histopathological features, fibrosis in testicular tissue, abnormalities in Sertoli cell, and up-regulation in caspase-3 were observed. Conversely, GA administration successfully reversed these issues.

Conclusion

The ability of GA to counteract toxicological molecular targets in Tz-exposed testes is believed to be achieved through the restoration of oxidant/antioxidant balance and the prevention of the apoptotic cascade.

Background

Synthetic colorants play a significant role in the food processing industry, serving consumer preferences and enhancing the appeal of food products. They provide color to food items, making them more visually appealing and easily recognizable (Dey & Nagababu, 2022). However, recent attention has focused on the potential side effects of food additives, particularly azo dyes, due to their widespread use driven by affordability and long-term stability (Kaya et al., 2021). Tartrazine (Tz), a commonly used member of this group, is found in various products such as beverages, fruit juices, chocolates, cookies, chewing gum, jams, candies, ice cream, sauces, and mustard (Zingue et al., 2021). A debate arises concerning safety window of Tz as some peer-reviewed articles confirmed lack of adverse effects (EEDAP et al., 2016; Pestana et al., 2010), while others presented an opposite viewpoint (Abd-Elhakim et al., 2019; Altinoz et al., 2021; Ismail & Rashed, 2022). Tz has been associated with various harmful consequences in humans, including attention deficit hyperactivity disorder (Miller et al., 2022), inflammatory responses (Leo et al., 2018), and allergic reactions (Ardern, 1996). Studies in rodents have also shown evidence of liver toxicity (Hashem et al., 2019), inflammatory effects (Meyer et al., 2017), and learning/memory defects (Gao et al., 2011) linked to Tz consumption.

Metabolic derivatives of Tz, such as sulphanilic acid and aminopyrazolone, generate excessive reactive oxidants that trigger lipid peroxidation, disrupt enzymatic antioxidative defenses, and damage subcellular structures, ultimately leading to cell death (Bhatt et al., 2018; Morales et al., 2004). The unique physiological characteristics of the testes, including a high rate of cell proliferation, oxygen consumption, and a rich content of unsaturated fats, make them particularly susceptible to peroxidative damage caused by Tz exposure (Asadi et al., 2017). According to the available literature, the not yet fully-researched testicular toxicity of Tz could be mediated by hindering spermatogenesis, testosterone output and testicular redox network and activating lipid peroxidation cascade (Ara et al., 2022; Boussada et al., 2017; El Rabey et al., 2019). An increase in the expression of testicular protein kinase 1, a gene involved in the meiotic phase of spermatogenesis, is implicated in testicular damage associated with Tz exposure. The disturbances in reproductive hormonal axis were mediated by alterations in fertility hormone receptor gene expressions (Wopara et al., 2021). Elevated levels of caspase-3 transcripts and reduced levels of Bcl-2 transcripts after Tz intervention may initiate cellular apoptosis in the testes Essawy et al., 2024). The down-regulation in expressions of the genes coding for catalase, glutathione peroxidase-1, glutathione-S-transferase, and nuclear factor erythroid 2-related factor 2 could be incriminated in the pro-oxidative effect of Tz on testicular microenvironment (Eteng et al., 2021).

Challenges such as low stability, weak coloring strength, interactions with food ingredients, and the inability to achieve desired hues hinder the substitution of synthetic colorants with natural alternatives (Sigurdson et al., 2017). Hence, incorporating natural protective agents into the diet to mitigate the harmful effects of synthetic food colorants offers a preferable option, with gallic acid (GA) standing out as a promising nutritional supplement. GA is a group of phenolic compounds, also identified as 3,4,5-trihydroxybenzoic acid, is a naturally produced secondary metabolite present in a range of plants, vegetables, nuts, and fruits (Govea‑Salas et al., 2016). GA, when utilized as a supplement, has the potential to inhibit the rancidity and deterioration of fats and oils in a variety of consumable products, including condiments, confectionery, beverages, and baked and fried foods (Kosuru et al., 2018). GA counteracts nephrotoxicity, hepatotoxicity, neurotoxicity, pulmonary fibrosis, myocardial injury, and metabolic disorders (Kahkeshani et al., 2019). It also known for its ability to enhance the pituitary–gonadal axis, maintain testicular redox balance, and preserve histo-architecture through its antioxidant, anti-apoptotic, and cytoprotective properties in various experimental models of testicular dysfunction (Hosseinzadeh et al., 2022; Olukole et al., 2020; Owumi et al., 2022). Nevertheless, there is a shortage in data regarding the plausible role of GA in fighting the Tz-induced testicular abnormalities. Thus, this study aims to address this gap in knowledge, considering the similarity in the toxicokinetic pathways of Tz in both human beings and laboratory animals (Elhkim et al., 2007), providing a hope to clinically apply the findings and increase the safety limit of artificial azo dyes.

Methods

Chemicals

Gallic acid (GA) and tartrazine (Tz) were purchased from the Indian companies Sd. Fine Chem. Ltd and Marine Chemicals Company, respectively. All other chemicals and reagents used were of the highest available commercial purity.

Experimental protocol

In this investigation, 18 adult male Wistar rats weighing 180 ± 30 g were employed. Animals were obtained from the Animal House of Faculty of Pharmacy, Cairo University, Egypt. Animals were housed in stainless steel cages at room temperature under the identical laboratory conditions of temperature (25 ± 2 °C) and 12 h light/12 h dark cycle. They were fed water and a regular commercial pellet meal during the research period. Rats were allocated randomly and equally into three groups (10 per group) for 30 days. Control group received no treatment. Tz group was daily administrated Tz at a dose of 30 mg/kg BW (Amin et al., 2010). Tz + GA group was administrated GA at a dose of 200 mg/kg BW (Karimi-Khouzani et al., 2017) in association with the above-mentioned dose of Tz. Both Tz and GA were supplemented orally once daily by a stomach tube. A single animal was used as an experimental unit. To maintain blinding throughout the experiment, the animal care technician was unaware of the group distribution. The primary outcome measure in this experiment was the changes in pituitary–gonadal hormones following the interventions.

Sample collection

At the end of the experiment, overnight fasted rats were anaesthetized by sodium thiopental, and blood was promptly drawn from each rat’s orbital sinus by heparinized microcapillary tubes. The blood samples were collected in EDTA tubes and centrifuged at 3000 rpm for 15 min to obtain plasma. To gather tissue samples, rats were killed through cervical dislocation. The testis was milked out of the incision site and promptly exposed by cutting the tunica vaginalis during the orchidectomy, which was done using an open castration approach through a midline incision. 0.5 g of testis was homogenate in 5 ml (0.1 M) phosphate buffer (pH 7.4) using a homogenizer (IKA Yellow line DI 18 Disperser, Germany). The homogenates were centrifuged for 1 h at 4 °C at 6000 rpm, and the supernatant cytosols were frozen at − 20 °C. Plasma and tissue hemolysates were kept at − 20 °C for the biochemical evaluations.

Biochemical analysis

Luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone, and 17β-estradiol (E2) were measured by ELISA kits (Catalog No. LH231F, FS232F, TE373S, and ES380S, respectively) according to manufacturer’s instructions (Calbiotech Inc, El Cajon, USA). Malondialdehyde (MDA) was estimated according to the method of (Ohkawa et al., 1979) using tetramethoxypropane as an external standard. Nitric oxide (NO) was measured based on an earlier procedure (Ding et al., 1988) which based on conversion of phosphoric acid to a diazonium salt by reaction with nitrite in acid solution. The diazonium salt is then coupled to naphthylene diamine dihydrochloride forming an azo dye that can be spectrophotometrically quantitated. Total antioxidant capacity (TAC) was assessed according to the manufacturer’s instructions using commercial colorimetric kit (Catalog number: TA 2513) provided by Egyptian Company for Biotechnology, Cairo, Egypt. Glutathione (GSH) content was estimated using the method of (Beutler et al., 1963).

Histopathology

Testis tissue samples were fixed in 10% neutral buffered formalin, dehydrated with ascending alcohol grades, cleared with xylene, and embedded in paraffin. Tissue sections, 5 microns thick, were prepared and stained with hematoxylin and eosin (H&E) for general histological assessment and Sirius red staining to detect fibrosis (collagen I & III) (Banchroft et al., 1996).

Morphometric analysis

After histological testes tissue was processed, photographs were taken under an objective lens magnification of 40 × using a digital camera connected to a light microscope (Leitz Dialux 20 Microscope). This analysis was done with the computerized image analysis software system Image J. Spatial calibration by object micrometer was performed before each analysis. Five pictures were selected from each animal in each group. The following morphometric parameters were measured: mean value of the intensity of collagen I and III (red area) by Sirius red stain (%) and mean value of the intensity of caspase-3 immunostaining expression (%).

Transmission electron microscope

The samples were fixed in 5% cold glutaraldehyde for 24–48 h immediately after animal necropsy. Samples were then washed 3–4 times with cacodylate buffer (pH 7.2) for 20 min each, post-fixed with 1% O₄S₄ for 2 h, and then washed 4 times with the same buffer. Dehydration with ascending alcohol content (30, 50, 70, 90, and 100% for 2 h) was done (Bozzola & Russell, 1999), followed by embedding in epon mixture. Semi-thin sections (0.5–1 microns thick) were cut from embedded blocks using an LKB ultramicrotome. A Leica AG ultramicrotome was used to slice the tissue into ultra-thin sections (500–700 Å thick), and contrasted with uranyl acetate and lead citrate. The tissues were orientated and shot using a SC30 Olympus camera. CCD digital camera Model XR-41 and JEM 100 CXII electron microscope at 80 kV were used to inspect and take pictures. The program Photo Filter 6.3.2 we digitally colored the TEM pictures in order to identify various cell and structural types.

Immunohistochemistry of caspase-3

Paraffin sections of the testis were cleared in xylene, rehydrated in graded ethanol, immersed in water for 5–10 min, and incubated in 0.3% H₂O₂ and 70% methanol for 20 min to inhibit endogenous peroxidase activity. The specimens were then rinsed three times for five minutes in phosphate buffer saline (PBS), and epitopes were disguised by boiling in citrate buffer (pH 6.0) for 10–15 min. After rinsing in PBS, the specimens were blocked for 30–60 min in 3% bovine serum albumin in PBS, and were incubated with primary antibody against caspase-3 in 0.1% bovine serum albumin in PBS overnight at 4°C in a humidified chamber. The samples were then rinsed in PBS and incubated with 7.5 g/mL of biotinylated secondary antibody in 0.1% bovine serum albumin in PBS for 1 h at room temperature, followed by avidin–biotin amplification for 30 min, and were developed with 3,3- diaminobenzidine peroxidase substrate. Sections were counter-stained with Mayer hematoxylin for three minutes and mounted. Negative controls were obtained by substituting the primary antibody with PBS (Kazi et al., 2003).

Statistical analysis

The data were expressed as means ± standard error of the mean. Statistical variances between groups were assessed using one-way ANOVA, followed by the Duncan post-test. Prior to analysis, normality of the data was evaluated using the Shapiro–Wilk test, and homogeneity of variances was confirmed to ensure that the data met the assumptions of normal distribution and equal variances. Statistical analysis was performed using SPSS software version 16 (SPSS, Richmond, VA, USA). A difference was regarded as statistically significant when p < 0.05.

Results

Biochemical findings

The Tz group exhibited significant perturbations in the pituitary–gonadal axis, characterized by a significant decrease in LH, FSH, testosterone, and E2 levels compared to the control group. Although these reproductive outcomes were significantly increased in the Tz + GA group, they remained below the levels observed in the control group, except for E2, which showed no significant difference from the control group. Tz-induced testicular toxicity led to disturbances in plasma redox balance, evident by a significant increase in MDA and NO levels, along with a significant decrease in TAC and GSH levels. Co-administration of GA with Tz effectively restored the redox balance by normalizing these parameters (Table 1).

Table 1 Effects of gallic acid on the plasma pituitary–gonadal hormones and redox parameters of tartrazine-intoxicated rats

Histopathological findings

The lesion score of the histopathological results of the testes in all studied groups were summarized in Table 2. Histological examination of the testes in the Tz group revealed interstitial edema containing a foamy, proteinaceous fluid and congested blood vessels. Some sections showed dilated seminiferous tubules (ST) and arrested spermatogenesis. Irregularity in shape of some ST and large area of proliferated Leydig cells was also prominent (Fig. 1). In contrast, the Tz + GA group showed relatively normal seminiferous tubules with complete spermatogenesis and interstitial edema in some sections (Fig. 2A–C). The control group displayed normal appearance of seminiferous tubules with different stages of spermatogenesis and normal interstitial tissues (Fig. 2D, E).

Table 2 The scores of histological lesions and immunohistochemical findings in the testicular tissue of various experimental groups

Fig. 1

Photomicrographs of rat testis sections of Tz group (A–H) stained by H&E showing; interstitial edema (stars), congestion of the blood vessels (black arrow), proliferation of Leydig cells (yellow arrows), seminiferous tubules are dilated with arrest of spermatogenesis (ST), and Irregularity in shape of some seminiferous tubules (head arrows). H&E

Fig. 2

Photomicrographs of rat testis sections stained by H&E showing; A–C Tz + GA group: slightly normal seminiferous tubules with complete spermatogenesis (ST) and interstitial edema (star). D, E Control group: normal appearance of seminiferous tubules with complete stages of spermatogenesis (ST). H&E

Sirius red staining for fibrosis confirmed the presence of a large amount of fibrous tissues in the interstitial tissues, around blood vessels, and around seminiferous tubules in the Tz group (Fig. 3A), while fibrosis was reduced in the Tz + GA group (Fig. 3B). The control group showed a mild amount of fibrous tissues around blood vessels and seminiferous tubules (Fig. 3C). Statistically, there were significant differences between the groups as shown in Fig. 3D.

Fig. 3

Photomicrographs of rat testis sections stained by Sirius red showing; A Tz group: increase amount of red fibrous tissue. B Tz + GA group: moderate amount of fibrous tissue. C Control group: mild amount of red fibrous tissue. Around blood vessels (head arrow), around seminiferous tubules (arrows), and in the interstitial tissues (stars). D The mean value of the intensity of Sirius red stain for collagen I & III in control and different treated groups. ^a−c different letters indicate significant differences at p < 0.05 between the groups

Ultrastructural findings

Examination of seminiferous tubules in the Tz group revealed the presence of Sertoli cells with necrosis in their nuclei and multiple large vacuoles. Hemorrhages were observed in the interstitial spaces (Fig. 4A, B). The Tz and Tz + GA groups showed normal appearance of spermatocytes (Fig. 4C). The control group exhibited typical features of spermatocytes and spermatids (Fig. 4D).

Fig. 4

Transmission electron micrograph: A, B Tz group showing seminiferous tubules with Sertoli cells only (S), vacuolation (V) and interstitial hemorrhages (red arrows). C Tz + GA group showing normal appearance of spermatocytes. D Control group showing normal appearance of spermatocytes (SC) and spermatids (SP)

Immunohistochemistry of caspase-3

The Tz group showed severe expression of caspase-3 in intracytoplasmic testicular cells and in the interstitial tissue cells compared to the control (Fig. 5A). The positive reaction of caspase-3 in the interstitial tissue cells was obvious in the Tz + GA group (Fig. 5B). The control group revealed a slight positive caspase-3 expression in very few interstitial tissue cells (Fig. 5C). Statistical analysis showed a significant difference in caspase-3 expression between the groups (Fig. 5D).

Fig. 5

Immunohistochemical staining of caspase-3 expression in rat testis: A Tz group showing strong positive caspase-3 immunostaining (deep brown) appeared in intracytoplasmic of seminiferous tubules (arrow), and in the interstitial tissue cells (star). B Tz + GA group showing positive expression in the interstitial tissues only and negative expression in seminiferous tubules. C Control group showing slightly positive caspase-3 immunostaining in the interstitial tissues only. D The mean value of caspase-3 immunostaining expression in control and different treated groups. ^a−cDifferent letters indicate significant differences at p < 0.05 between the groups

Discussion

The dysregulation in the pituitary–gonadal axis following the exposure to Tz is consistent with previous reports (Elewa et al., 2019). Decreased expression levels of testicular steroidogenic enzymes and androgen and gonadotropic receptors, and disruption of the reproductive axis by azo dye colorants (Abbas & Al-Hamadawi, 2019; Alsudani & Alhamadawi, 2020; Montaser et al., 2018) are suggested to be explanatory factors. Low LH output impairs the ability of Leydig cells to synthesize testosterone, as it is needed to translocate cholesterol into mitochondria (Zirkin & Papadopoulos, 2018). The drop in testosterone could happen due to the detrimental influence of extra release of reactive oxygen species and depletion of antioxidant stock on Leydig cells (Mohamed et al., 2017). The improvement in reproductive hormones following GA supplementation in concurrent UA exposure agrees with that found in zearalenone-mediated testicular defects in rats (Owumi et al., 2022). This outcome is attributed to increased mRNA levels of steroidogenic acute regulatory protein (Dai et al., 2021).

The disturbance in redox equilibrium in Tz-exposed rats is consistent with that observed in serum, kidney, and liver homogenate in previous reports (El Rabey et al., 2019). The aromatic amines, which are released following the biotransformation of Tz by intestinal microbiota, produce reactive oxidants when interacting with dietary nitrogenous compounds (Moutinho et al., 2007). In response to the surplus formation of free radicals, the antioxidant defense capacity of the body started to be consumed to stop their adverse impacts, while MDA accumulated as a result of lipid peroxidation caused by their action on cellular membrane lipids (Amin et al., 2010). The reduction in plasma GSH content could be attributed to the increased activity of glutathione peroxidase which consumes GSH reserve during reduction of hydrogen peroxide and lipid peroxides (Bhatt et al., 2018). Down-regulation of nuclear factor erythroid 2-related factor 2, a main regulator to a variety of antioxidant response element–dependent genes, can participate in the depletion of plasma TAC and GSH levels (Akintoye et al., 2022). Overgeneration of reactive oxygen species is responsible for lipid peroxidation, and genomic fragmentation in the reproductive system, and germ cell death (Agarwal et al., 2020). Thus, interrupting the propagation of free radicals can limit autoxidation and subsequently block the lipid peroxidation cascade. Accordingly, we used GA to counteract the Tz-linked redox imbalance owing to its free radicals-scavenging properties and its ability to up-regulate antioxidant gene expression (Gao et al., 2019; Ojeaburu & Oriakhi, 2021). Several scholarly articles confirmed the testicular protective effect of GA against chemotoxicants secondary to its redox-stabilizing action (Abarikwu et al., 2022; Owumi et al., 2020).

Similar to a previous finding (Khayyat et al., 2017), the plasma NO of the Tz group elevated due to increased transcript abundance of endothelial NO synthase (Abd-Elhakim et al., 2018). The raised NO level in the Tz group inhibits testicular steroidogenesis and gonadotropin-releasing hormone-induced LH secretion (Chatterjee et al., 1997; Kostic et al., 2000). Dysfunction in sperm characteristics and a drop in gonadotropin output are the consequences of NO when presents in excess (Dutta & Sengupta, 2022). Also, it up-regulates the expression levels of caspase-3 and Bax and down-regulates the expression level of Bcl-2 (Zhou et al., 2019). The reduction in the protein expression level of inducible NO synthase (Mard et al., 2015) could explain the normalization of NO level in the Tz + GA group.

The histoarchitectural deteriorations in the testis of TZ-exposed rats are in harmony with the findings of (Elewa et al., 2019). These cytological responses could emerge from lipid peroxidation as metabolic byproducts of Tz elicit oxidative overloading (Bhatt et al., 2018). ST dilatation might be due to a reduction in E2 failing the efferent ducts to resorb ST fluid with possible compression and atrophy of germinal epithelium (Heuser et al., 2013). The arrest in spermatogenesis in the histological sections is similar to (Al-Seeni et al., 2018). By generating regulatory signals and nutritive supplies for preserving the growing germ cells, FSH both alone and in conjunction with either LH or testosterone influences the multiplication, development, and viability of the sustentacular Sertoli cells (Oduwole et al., 2018, 2021). Thus, the depletion of reproductive hormones creates an unfavorable biological environment in maintenance and driving the spermatogenic potential. Sertoli cells in rats were harmed by TZ, reducing their ability to support spermatogenesis (Mahfouz & Moussa, 2015). It also suppresses the proliferation of spermatogonia and differentiation of Sertoli cells (Elewa et al., 2019). As a zinc-chelating compound, Tz deprives the testis of the indispensable role of zinc in cell proliferation and growth, resulting in the suppression of spermatogenesis (Visweswaran & Krishnamoorthy, 2012). Fibrosis in the interstitial and perivascular spaces is corresponding to what occurred in colon mucosa (Altinoz et al., 2021) and hepatic tissue (Balta et al., 2019) of TZ-supplemented rats; owing to up-regulation of fibrotic markers collagen 1-α, tumor growth factor β-1, and fibronectin (Abd-Elhakim et al., 2019). Sertoli cell vacuolation in the testis of Tz-intoxicated rats is in line with previous findings by (Montaser et al., 2018). These vacuoles could be regarded as locations of germ cell loss after elimination by Sertoli cells (Eid et al., 2002). These vacuoles mostly include damaged mitochondria that have been trapped within autophagic vacuoles (Eid et al., 2013). The cell vacuolation can be explained by promoting cell death or disrupting the autophagosome maturation (Cayli et al., 2020). The presence of vacuoles in Sertoli cells could impair their functions and be associated with infertility (Cayli et al., 2020).

GA intervention restored the testicular histological features in Tz-intoxicated rats in line with its improving effect on busulfan- and phthalate-induced testicular abnormalities (Abarikwu et al., 2022; Hosseinzadeh et al., 2022). The androgenic, cytoprotective, and antioxidant potentials of GA are positively reflected on the histo-architectural profile. From another mechanistic viewpoint, GA motivates the electron transport chain and oxidative phosphorylation to energize the germ cells (Gungor et al., 2019).

Intense immuno-expression of caspase-3 in the testis of TZ-challenged rats is in corresponding to what observed by (Mahfouz & Moussa, 2015). The increase in apoptotic/survival ratio under Tz burden is due to a decrease in mRNA levels of BCl-2 and proliferating cell nuclear antigen, increase in mRNA levels of Bax, and failure in repairing DNA damage (Elewa et al., 2019; Essawy et al., 2023; Haridevamuthu et al., 2023). Inadequate testosterone production by Leydig cells is associated with mitochondrial abnormalities by blocking respiratory electron flux and subsequently exhausting energy stores, causing eventually apoptosis (Wang et al., 2015). By activation of the mitochondrial, death receptor, and endoplasmic reticulum apoptotic pathways, excessive production of free radicals is incriminated in the induction of programmed cell death (Redza-Dutordoir & Averill-Bates, 2016). Increased NO declines the probability of cell survival by favoring apoptosis at the expense of autophagy (Zhang et al., 2019). GSH depletion disrupts the mitochondrial membrane permeability leading to the induction of intrinsic apoptotic pathway (Malla et al., 2020). As gonadotrophic hormones act as anti-apoptotic effectors (Li et al., 2020; Wang et al., 2018a), thus the decrease in their output could be causatively linked to the shift in the cell programming toward death decisions.

According to our findings and consistent with the gastric ulcer rat model, GA exhibited anti-apoptotic potential by decreasing the protein expression of caspase-3 and Bax and increasing the protein expression of Bcl-2 (Zhou et al., 2020). By lowering the Bax/Bcl-2 ratio and mitochondrial depolarization, increasing ATP generation, and blocking mitogen-activated protein kinase and Fas death receptor-mediated apoptosis, the drop in NO exerts an anti-apoptotic impact (Wang et al., 2018b). Thus, the reduction in the elevated NO level by GA supplementation hindered the process of cell death. The transcript abundance of proliferating cell nuclear antigen, a nuclear protein that encodes crucial DNA replication proteins, is enhanced by GA (dos Santos Silva et al., 2023), resulting in a reaction that encourages cellular survival over suicidal signals. Similar to the glyoxal-induced renal fibrotic rat model (Yousuf & Vellaichamy, 2015), concurrent administration of GA ameliorated fibrosis by reducing the protein expression of collagen type I, fibronectin, connective tissue growth factor and matrix metalloproteinase (Jin et al., 2018; Yousuf & Vellaichamy, 2015).

Conclusions

Our findings confirmed the sensitivity of testis to Tz exposure on both biochemical and cytological levels. Thus, it is mandatory to increase public knowledge of the health risks associated with this dye. GA was an effective phytochemical strategy in fighting Tz-related testicular disorders by restoring reproductive hormonal balance and oxidant/antioxidant responses and dampening cellular death signals and histological lesions. Therefore, GA provides a relatively safe and cost-effective approach to antagonizing gonadotoxic agents. However, it’s important to acknowledge the limitations of our study, including the lack of assessment of lipid peroxidation biomarker levels, antioxidant activities in testicular tissue, and semen quality parameters. Further studies are strongly recommended to highlight the potential protective effects of GA against other health problems induced by Tz.